Formulations of 2-(4-chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamide

ABSTRACT

Provided herein are lyophilized formulations of 2-(4-chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamide or a stereisomer or mixture of stereisomers, pharmaceutically acceptable salt, tautomer, prodrug, solvate, hydrate, co-crystal, clathrate, or polymorph thereof. Methods of using the formulations and dosage forms for treating, managing, and/or preventing cancer are also provided herein.

1. CROSS REFERENCE TO RELATED APPLICATION

This application claims priority under 35 U.S.C. § 120 to, and is acontinuation of U.S. application Ser. No. 15/400,791 filed Jan. 6, 2017,currently allowed, which claims the benefit of the priority of U.S.Provisional Application No. 62/276,756, filed Jan. 8, 2016, thedisclosures of each which are incorporated herein by reference in theirentireties.

2. FIELD

Provided herein are formulations and dosage forms of2-(4-chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamideor a stereoisomer or a mixture of stereisomers, pharmaceuticallyacceptable salt, tautomer, prodrug, solvate, hydrate, co-crystal,clathrate, or polymorph thereof. Methods of using the formulations anddosage forms for treating, managing, and/or preventing cancer are alsoprovided herein.

3. BACKGROUND

Drug substances are usually administered as part of a formulation incombination with one or more other agents that serve varied andspecialized pharmaceutical functions. Dosage forms of various types maybe made through selective use of pharmaceutical excipients.Pharmaceutical excipients have various functions and contribute to thepharmaceutical formulations in many different ways, e.g.,solubilization, dilution, thickening, stabilization, preservation,coloring, flavoring, etc. The properties of pharmaceutical excipientsthat are considered when formulating an active drug substance includebioavailability, ease of manufacture, ease of administration, andstability of the dosage form. Due to the varying properties of theactive drug substance to be formulated, and cross-reactivity betweenexcipients, dosage forms typically require pharmaceutical excipientsthat are uniquely tailored to the active drug substance to achieveadvantageous physical and pharmaceutical properties.

Nevertheless, use of pharmaceutical excipients in formulating dosageforms can, in some instances, cause undesirable adverse reactions withthe active ingredient which manifest upon, for example, prolongedstorage or contact with water. Indeed, it is well known that theproperties of the final dosage form (e.g., its bioavailability andstability) are, for the most part, highly dependent on the excipientschosen, their concentration and interaction with both the activecompound and each other. Excipients are more than inert or inactiveingredients and must be selected to avoid undesirable cross-reactionwith active ingredients and other excipients in the formulation.Selecting compatible excipients is critical in formulating dosage formsto ensure the active ingredient is properly delivered and that thedosage form is a stable formulation.

2-(4-Chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamideor a stereisomer or mixture of stereisomers, pharmaceutically acceptablesalt, tautomer, prodrug, solvate, hydrate, co-crystal, clathrate, orpolymorph thereof has been shown to have anticancer activities. There isa need for formulations of2-(4-chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamideor a stereoisomer or mixture of stereoisomers, pharmaceuticallyacceptable salt, tautomer, prodrug, solvate, hydrate, co-crystal,clathrate, or polymorph thereof for treatment of cancer.

4. BRIEF SUMMARY

Provided herein are lyophilized formulations comprising2-(4-chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamide,or a stereisomer or mixture of stereisomers, pharmaceutically acceptablesalt, tautomer, prodrug, solvate, hydrate, co-crystal, clathrate, orpolymorph thereof (“Compound 1”) and a pharmaceutically acceptableexcipient. Compound 1 is described in U.S. Pat. No. 9,499,514 andInternational Publication No. WO 2016/007848, the disclosures of eachwhich are incorporated herein by reference in their entireties. In oneembodiment, Compound 1 is polymorph Form A, Form B, Form C, Form D, FormE or an amorphous form of2-(4-chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamide.In one embodiment, Compound 1 is polymorph Form C of2-(4-chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamide.The polymorphs of2-(4-chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamideare described herein and in U.S. provisional patent application No.62/276,750 filed on Jan. 8, 2016, entitled SOLID FORMS OF2-(4-CHLOROPHENYL)-N-((2-(2,6-DIOXOPIPERIDIN-3-YL)-1-OXOISOINDOLIN-5-YL)METHYL)-2,2-DIFLUOROACETAMIDE,AND THEIR PHARMACEUTICAL COMPOSITIONS AND USES”, the disclosure of whichis incorporated herein by reference in its entirety.

In certain embodiments, the lyophilized formulations provided hereincomprise a solid form of2-(4-chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamide.In certain embodiments, the lyophilized formulations provided hereincomprise an amorphous form of2-(4-chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamide.

In one aspect, the lyophilized formulations provided herein are suitablefor reconstitution with a suitable diluent to the appropriateconcentration prior to administration. In one embodiment, thelyophilized formulation is stable at room temperature. In oneembodiment, the lyophilized formulation is stable at room temperaturefor up to about 24 months. In one embodiment, the lyophilizedformulation is stable at room temperature for up to about 24 months, upto about 18 months, up to about 12 months, up to about 6 months, up toabout 3 months or up to about 1 month. In one embodiment, thelyophilized formulation is stable upon storage under acceleratedcondition of 40° C./75% RH for up to about 12 months, up to about 6months or up to about 3 months.

In one aspect, the lyophilized formulation provided herein is suitablefor reconstitution with an aqueous solution for intravenousadministrations. In one aspect, the lyophilized formulation providedherein is suitable for reconstitution with water. In one embodiment, thereconstituted aqueous solution is stable at room temperature for up toabout 24 hours upon reconsititution. In one embodiment, thereconstituted aqueous solution is stable at room temperature from about1-24, 2-20, 2-15, 2-10 hours upon reconsititution. In one embodiment,the reconstituted aqueous solution is stable at room temperature for upto about 20, 15, 12, 10, 8, 6, 4 or 2 hours upon reconsititution. Incertain embodiments, the lyophilized formulations upon reconstitutionhave a pH of about 4 to 5.

In certain embodiment, the lyophilized formulations provided hereincomprise Compound 1, a pH adjusting agent and a bulking agent.

In one embodiment, the lyophilized formulation provided herein comprisesabout 0.1-2% Compound 1, about 1-15% buffer and about 70-95% bulkingagent based on the total weight of the lyophilized formulation.

In another aspect provided herein is a lyophilized formulationcomprising Compound 1 in about 0.1 to about 2% based on the total weightof the lyophilized formulation. In still another aspect, provided hereinis a lyophilized formulation that comprises Compound 1 in an amount ofabout 0.1 mg to about 5 mg in a vial, for example, a 20 cc vial.

In one aspect, the formulations provided herein comprise a citratebuffer in an amount from about 5% to about 25% based on total weight ofthe lyophilized formulation. In one embodiment, the citrate buffercomprises anhydrous citric acid and anhydrous sodium citrate.

In one aspect, the bulking agent in the formulations provided hereincomprises Captisol®, mannitol or Kleptose®, for example, β-cyclodextrin,hydroxypropyl β-cyclodextrin and methylated β-cyclodextrin.

In certain embodiments, provided herein is a unit dosage form comprisinga lyophilized formulation, wherein the lyophilized formulation comprisesCompound 1, a buffer and a bulking agent.

In certain embodiments, provided herein is a container comprising alyophilized formulation provided herein. In one aspect, the container isa glass vial.

In certain embodiments, provided herein are methods of treating,preventing or ameliorating cancer, including solid tumors and bloodborne tumors, or one or more symptoms or causes thereof. In certainembodiments, provided herein are methods of preventing cancer, includingsolid tumors and blood borne tumors, or one or more symptoms or causesthereof. In certain embodiments, provided herein are methods ofameliorating cancer, including solid tumors and blood borne tumors, orone or more symptoms or causes thereof. In certain embodiments, theblood borne tumor is leukemia. In certain embodiments, methods providedherein encompass methods of treating various forms of leukemias such aschronic lymphocytic leukemia, chronic myeloid leukemia, acutelymphocytic leukemia, acute myeloid leukemia and acute myeloblasticleukemia. In certain embodiments, methods provided herein encompassmethods of preventing various forms of leukemias such as chroniclymphocytic leukemia, chronic myeloid leukemia, acute lymphocyticleukemia, acute myeloid leukemia and acute myeloblastic leukemia. Incertain embodiments, methods provided herein encompass methods ofmanaging various forms of leukemias such as chronic lymphocyticleukemia, chronic myeloid leukemia, acute lymphocytic leukemia, acutemyeloid leukemia and acute myeloblastic leukemia. The methods providedherein include treatment of leukemias that are relapsed, refractory orresistant. The methods provided herein include prevention of leukemiasthat are relapsed, refractory or resistant. The methods provided hereininclude management of leukemias that are relapsed, refractory orresistant. In one embodiment, methods provided herein encompass methodsof treating acute myeloid leukemia. In one embodiment, methods providedherein encompass methods of preventing acute myeloid leukemia. In oneembodiment, methods provided herein encompass methods of managing acutemyeloid leukemia. In one embodiment, methods provided herein encompassmethods of treating a myelodysplastic syndrome. In one embodiment,methods provided herein encompass methods of preventing amyelodysplastic syndrome. In one embodiment, methods provided hereinencompass methods of managing a myelodysplastic syndrome.

In one embodiment, provided herein are methods of treating acute myeloidleukemia by intravenous administration of a formulation comprisingCompound 1. In one embodiment, provided herein are methods of treating amyelodysplastic syndrome by intravenous administration of a formulationcomprising Compound 1.

In practicing the methods, compositions containing therapeuticallyeffective concentrations of Compound 1 are administered to an individualexhibiting the symptoms of the disease or disorder to be treated. Theamounts are effective to ameliorate or eliminate one or more symptoms ofthe disease or disorder.

Further provided is a pharmaceutical pack or kit comprising one or morecontainers filled with one or more of the ingredients of thepharmaceutical compositions. Optionally associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use of sale for human administration. The pack or kit canbe labeled with information regarding mode of administration, sequenceof drug administration (e.g., separately, sequentially or concurrently),or the like.

These and other aspects of the subject matter described herein willbecome evident upon reference to the following detailed description.

5. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an X-ray powder diffractogram stack plot of Forms A, B,C, D, and E of Compound 1.

FIG. 2 depicts an X-ray powder diffractogram (XRPD) plot of Form A ofCompound 1.

FIG. 3 depicts a SEM image of Form A of Compound 1.

FIG. 4 depicts a thermogravimetrical analysis (TGA) plot of Form A ofCompound 1.

FIG. 5 depicts a differential scanning calorimetry (DSC) thermogram plotof Form A of Compound 1.

FIG. 6 provides a dynamic vapor sorption (DVS) isotherm plot of Form Aof Compound 1.

FIG. 7 provides a ¹H NMR spectrum of Form A of Compound 1.

FIG. 8 depicts the comparison of the X-ray powder diffractogram plots ofForm A of Compound 1 before (a) and after (b) compression.

FIG. 9 depicts an XRPD plot of Form B of Compound 1.

FIG. 10 depicts a SEM image of Form B of Compound 1.

FIG. 11 depicts a TGA thermogram plot of Form B of Compound 1.

FIG. 12 depicts a DSC thermogram plot of Form B of Compound 1.

FIG. 13 provides a DVS isothterm plot of Form B of Compound 1.

FIG. 14 provides a ¹H NMR spectrum of Form B of Compound 1.

FIG. 15 depicts the comparison of the X-ray powder diffractogram plotsof Form B of Compound 1 before (a) and after (b) compression.

FIG. 16 depicts an XRPD plot of Form C of Compound 1.

FIG. 17 depicts a SEM image of Form C of Compound 1.

FIG. 18 depicts a TGA thermogram plot of Form C of Compound 1.

FIG. 19 depicts a DSC thermogram of Form C of Compound 1.

FIG. 20 provides a DVS isotherm plot of Form C of Compound 1.

FIG. 21 provides a ¹H NMR spectrum of Form C of Compound 1.

FIG. 22 depicts the comparison of the X-ray powder diffractogram plotsof Form C of Compound 1 before (a) and after (b) compression.

FIG. 23 depicts an XRPD plot of Form D of Compound 1.

FIG. 24 depicts a TGA thermogram plot of Form D of Compound 1.

FIG. 25 depicts an XRPD plot of Form E of Compound 1.

FIG. 26 depicts a TGA thermogram plot of Form E of Compound 1.

FIG. 27 depicts the modulated DSC thermogramplot of amorphous Compound1.

FIG. 28 depicts an XRPD plot of amorphous Compound 1.

FIG. 29 depicts a ¹HNMR spectrum of amorphous Compound 1.

FIGS. 30A, 30B and 30C. Illustrate XRPD profiles of the lyophilizedformulations in the second screen.

FIG. 31. Illustrates temperature profile of the final lyophilizationprocess.

FIG. 32. Illustrates a process diagram of Compound 1 formulation.

6. DETAILED DESCRIPTION 6.1 Definitions

Generally, the nomenclature used herein and the laboratory procedures inorganic chemistry, medicinal chemistry, and pharmacology describedherein are those well known and commonly employed in the art. Unlessdefined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this disclosure belongs.

The term Compound 1 refersto“2-(4-chlorophenyl)-N-((2-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamide”having the structure:

and its stereoisomers or mixture of stereoisomers, pharmaceuticallyacceptable salts, tautomers, prodrugs, solvates, hydrates, co-crystals,clathrates, or polymorphs thereof. In certain embodiments, Compound 1refers to2-(4-chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamideand its tautomers. In certain embodiments, Compound 1 refers to apolymorph of2-(4-chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamide.In certain embodiments, Compound 1 refers to polymorph Form C of2-(4-chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamide.In one embodiment, the stereoisomer is an enantiomer.

As used herein, and unless otherwise specified, the term “lyophilize”refers to the process of isolating a solid substance from solutionand/or removal of solvent. In some embodiments, this may be achieved byvarious techniques known to one of skill in the art, including, forexample, evaporation (e.g., under vacuum, for example by freeze drying,and/or freezing the solution and vaporizing the frozen solvent undervacuum or reduced pressure conditions, etc.). In some embodiments, thesolution contains a cosolvent.

As used herein, the term “cosolvent” refers to a solvent that aids thesolubilization of an active agent in water during the manufacturing of alyophilized formulation provided herein. The cosolvent can be a solventthat also provides sufficient stability of the intermediate formulationduring manufacture. The cosolvent can also be removed from thelyophilized formulation, or reduced to an acceptable level, duringmanufacture. Examples of cosolvents include acetonitrile, chloroform,tert-butanol, dimethylacetamide, methanol, tetrahydrofuran, acetic acid,acetone, anisole, butanol, butyl acetate, tert-butylmethyl ether,ethanol, ethyl acetate, ethyl ether, ethyl formate, heptanes, isobutylacetate, isopropyl acetate, methyl acetate, 3-methyl-butanol,methylethyl ketone, methylisobutyl ketone, 2-methyl-1-propanol, pentane,1-pentanol, 1-propanol, 2-propanol, and propyl acetate.

As used herein, and unless otherwise specified, the term “parenteral”includes subcutaneous, intravenous, intramuscular, intra-artricular,intra-synovial, intrasternal, intrathecal, intrahepatic, intralesionaland intracranial injection or infusion techniques.

As used herein, and unless otherwise specified, the term “substantiallyfree of” means containing no more than an insignificant amount. In someembodiments, a composition or preparation is “substantially free of” arecited element if it contains less than 5%, 4%, 3%, 2%, or 1%, byweight of the element. In some embodiments, the composition orpreparation contains less than 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%,0.2%, 0.1% or less of the recited element. In some embodiments, thecomposition or preparation contains an undetectable amount of therecited element.

As used herein, “reconstituted aqueous solution” or “reconstitutedaqueous composition” or “reconstituted aqueous formulation” refers to anaqueous solution obtained by dissolving a lyophilized compositionprovided herein in an aqueous solvent.

The term “aqueous diluent” used herein refers to an aqueous liquidcapable of being included in a parenteral formulation. Such aqueousdiluents can include, for example, saline or dextrose if desired, aswell as any of the known ancillary preservatives or excipients commonlyfound as part of parenteral formulations. Exemplary aqueous diluentsinclude water, 5% dextrose solution, and the like.

As used herein, and unless otherwise specified, the expression “unitdose” refers to a physically discrete unit of a formulation appropriatefor a subject to be treated (e.g., for a single dose); each unitcontaining a predetermined quantity of an active agent selected toproduce a desired therapeutic effect (it being understood that multipledoses may be required to achieve a desired or optimum effect),optionally together with a pharmaceutically acceptable carrier, whichmay be provided in a predetermined amount. The unit dose may be, forexample, a volume of liquid (e.g., an acceptable carrier) containing apredetermined quantity of one or more therapeutic agents, apredetermined amount of one or more therapeutic agents in solid form, asustained release formulation or drug delivery device containing apredetermined amount of one or more therapeutic agents, etc. It will beappreciated that a unit dose may contain a variety of components inaddition to the therapeutic agent(s). For example, acceptable carriers(e.g., pharmaceutically acceptable carriers), diluents, stabilizers,buffers, preservatives, etc., may be included as described infra. Itwill be understood, however, that the total daily usage of a formulationof the present disclosure will be decided by the attending physicianwithin the scope of sound medical judgment. The specific effective doselevel for any particular subject or organism may depend upon a varietyof factors including the disorder being treated and the severity of thedisorder; activity of specific active compound employed; specificcomposition employed; age, body weight, general health, sex and diet ofthe subject; time of administration, and rate of excretion of thespecific active compound employed; duration of the treatment; drugsand/or additional therapies used in combination or coincidental withspecific compound(s) employed, and like factors well known in themedical arts.

As used herein, the term “solid form” refers a crystal form or anamorphous form or a mixture thereof of2-(4-chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamideor a stereisomer or mixture of stereisomers, pharmaceutically acceptablesalt, tautomer, prodrug, solvate, hydrate, co-crystal, clathrate, orpolymorph thereof.

As used herein, unless otherwise specified, the term “pharmaceuticallyacceptable salt(s),” as used herein includes, but is not limited to,salts of acidic or basic moieties of compounds described herein (e.g.,Compound 1). Basic moieties are capable of forming a wide variety ofsalts with various inorganic and organic acids. The acids that can beused to prepare pharmaceutically acceptable acid addition salts of suchbasic compounds are those that form non-toxic acid addition salts, e.g.,salts containing pharmacologically acceptable anions. Suitable organicacids include, but are not limited to, maleic, fumaric, benzoic,ascorbic, succinic, acetic, formic, oxalic, propionic, tartaric,salicylic, citric, gluconic, lactic, mandelic, cinnamic, oleic, tannic,aspartic, stearic, palmitic, glycolic, glutamic, gluconic, glucaronic,saccharic, isonicotinic, methanesulfonic, ethanesulfonic,p-toluenesulfonic, benzenesulfonic acids, or pamoic (e.g.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate) acids. Suitable inorganicacids include, but are not limited to, hydrochloric, hydrobromic,hydroiodic, sulfuric, phosphoric, or nitric acids. Compounds thatinclude an amine moiety can form pharmaceutically acceptable salts withvarious amino acids, in addition to the acids mentioned above. Chemicalmoieties that are acidic in nature are capable of forming base saltswith various pharmacologically acceptable cations. Examples of suchsalts are alkali metal or alkaline earth metal salts and, particularly,calcium, magnesium, sodium, lithium, zinc, potassium, or iron salts.

As used herein, and unless otherwise specified, the term “solvate” meansa compound provided herein or a salt thereof that further includes astoichiometric or non-stoichiometric amount of solvent bound bynon-covalent intermolecular forces. Where the solvent is water, thesolvate is a hydrate.

As used herein and unless otherwise indicated, the term “prodrug” meansa derivative of a compound that can hydrolyze, oxidize, or otherwisereact under biological conditions (in-vitro or in-vivo) to provide thecompound. Examples of prodrugs include, but are not limited to,derivatives of compounds described herein (e.g., Compound 1) thatinclude biohydrolyzable moieties such as biohydrolyzable amides,biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzablecarbonates, biohydrolyzable ureides, and biohydrolyzable phosphateanalogues. Other examples of prodrugs include derivatives of compoundsdescribed herein (e.g., Compound 1) that include NO, NO₂, ONO, or ONO₂moieties.

A “pharmaceutically acceptable excipient,” refers to a substance thataids the administration of an active agent to a subject by for examplemodifying the stability of an active agent or modifying the absorptionby a subject upon administration. A pharmaceutically acceptableexcipient typically has no significant adverse toxicological effect onthe patient. Examples of pharmaceutically acceptable excipients include,for example, water, NaCl (including salt solutions), normal salinesolutions, sucrose, glucose, bulking agents, buffers, binders, fillers,disintegrants, lubricants, coatings, sweeteners, flavors, alcohols,oils, gelatins, carbohydrates such as amylose or starch, fatty acidesters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, andthe like. One of skill in the art will recognize that otherpharmaceutical excipients known in the art are useful in the presentinvention and include those listed in for example the Handbook ofPharmaceutical Excipients, Rowe R. C., Shesky P. J., and Quinn M. E.,6^(th) Ed., The Pharmaceutical Press, RPS Publishing (2009). The terms“bulking agent”, and “buffer” are used in accordance with the plain andordinary meaning within the art.

As used herein, “administer” or “administration” refers to the act ofphysically delivering a substance as it exists outside the body into asubject. Administration includes all forms known in the art fordelivering therapeutic agents, including but not limited to topical,mucosal, injections, intradermal, intravenous, intramuscular delivery orother method of physical delivery described herein or known in the art(e.g., implantation of a slow-release device, such as a mini-osmoticpump to a subject; liposomal formulations; buccal; sublingual; palatal;gingival; nasal; vaginal; rectal; intra-arteriole; intraperitoneal;intraventricular; intracranial; or transdermal).

“Anti-cancer agents” refer to anti-metabolites (e.g., 5-fluoro-uracil,methotrexate, fludarabine), antimicrotubule agents (e.g., vincaalkaloids such as vincristine, vinblastine; taxanes such as paclitaxel,docetaxel), alkylating agents (e.g., cyclophosphamide, melphalan,carmustine, nitrosoureas such as bischloroethylnitrosurea andhydroxyurea), platinum agents (e.g. cisplatin, carboplatin, oxaliplatin,JM-216 or satraplatin, CI-973), anthracyclines (e.g., doxorubicin,daunorubicin), antitumor antibiotics (e.g., mitomycin, idarubicin,adriamycin, daunomycin), topoisomerase inhibitors (e.g., etoposide,camptothecins), anti-angiogenesis agents (e.g. Sutent® and Bevacizumab)or any other cytotoxic agents, (estramustine phosphate, prednimustine),hormones or hormone agonists, antagonists, partial agonists or partialantagonists, kinase inhibitors, checkpoint inhibitors, and radiationtreatment.

By “co-administer” it is meant that a composition described herein isadministered at the same time, just prior to, or just after theadministration of one or more additional therapeutic compositions,including for example an anti-cancer agent. Co-administration is meantto include simultaneous or sequential administration of compoundsindividually or in combination (more than one compound or agent).Co-administration includes administering two active agentssimultaneously, approximately simultaneously (e.g., within about 1, 5,10, 15, 20, or 30 minutes of each other), or sequentially in any order.Thus, co-administration can include administering one active agent (e.g.a compound described herein) within 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20,or 24 hours of a second active agent. Co-administration can also beaccomplished by co-formulation, e.g., preparing a single dosage formincluding both active agents. The active agents can be formulatedseparately. In such instances, the active agents are admixed andincluded together in the final form of the dosage unit. Alternatively,co-administration as described herein can include administering twoseparate unit dosage forms of at least two separate active agents (e.g.,Compound 1 and a second active agent described herein).

As used herein, the term “daily” is intended to mean that a therapeuticcompound, such as Compound 1, is administered once or more than onceeach day for a period of time. The term “continuous” is intended to meanthat a therapeutic compound, such as Compound 1, is administered dailyfor an uninterrupted period of at least 10 days to 52 weeks. The term“intermittent” or “intermittently” as used herein is intended to meanstopping and starting at either regular or irregular intervals. Forexample, intermittent administration of Compound 1 is administration forone to six days per week, administration in cycles (e.g., dailyadministration for one to ten consecutive days of a 28 day cycle, then arest period with no administration for rest of the 28 day cycle or dailyadministration for two to eight consecutive weeks, then a rest periodwith no administration for up to one week), or administration onalternate days. The term “cycling” as used herein is intended to meanthat a therapeutic compound, such as Compound 1, is administered dailyor continuously but with a rest period.

An “effective amount” is an amount sufficient to achieve the effect forwhich it is administered (e.g., treat a disease or reduce one or moresymptoms of a disease or condition). Thus, administration of an “amount”of a compound described herein to a subject refers to administration of“an amount effective,” to achieve the desired therapeutic result. A“therapeutically effective amount” of a compound described herein forpurposes herein is thus determined by such considerations as are knownin the art. The term “therapeutically effective amount” of a compositiondescribed herein refers to the amount of the composition that, whenadministered, is sufficient to treat one or more of the symptoms of adisease described herein (e.g., AML). Administration of a compounddescribed herein can be determined according to factors such as, forexample, the disease state, age, sex, and weight of the individual. Atherapeutically effective amount also refers to any toxic or detrimentaleffects of Compound 1 are outweighed by the therapeutically beneficialeffects.

As used herein the terms “treat,” “treating” and “treatment” contemplatean action that occurs while a subject is suspected, diagnosed orsuffering from a disease described herein (e.g., leukemia, including AMLor MDS), which reduces the severity or symptoms of the disease, orretards or slows the progression or symptoms of the disease.

The terms “subject,” “patient,” “subject in need thereof,” and “patientin need thereof” are herein used interchangeably and refer to a livingorganism suffering from one or more of the diseases described herein(e.g., AML or MDS) that can be treated by administration of acomposition described herein. Non-limiting examples of organisms includehumans, other mammals, bovines, rats, mice, dogs, monkeys, goat, sheep,cows, deer, and other non-mammalian animals. In embodiments, a subjectis human. A human subject can be between the ages of about 1 year old toabout 100 years old. In embodiments, subjects herein can becharacterized by the disease being treated (e.g., a “AML subject”, a“cancer subject”, an “MDS subject” or a “leukemia subject”).

As used herein, and unless otherwise specified, the terms “prevent,”“preventing” and “prevention” refer to the prevention of the onset,recurrence or spread of a disease or disorder, or of one or moresymptoms thereof. The terms “prevent,” “preventing” and “prevention”contemplate an action that occurs before a patient begins to suffer fromthe specified disease or disorder or symptoms thereof, which inhibits orreduces the severity of the disease or disorder.

As used herein, and unless otherwise indicated, the terms “manage,”“managing” and “management” encompass preventing the recurrence of thespecified disease or disorder in a patient who has already suffered fromthe disease or disorder, or lengthening the time that a patient who hassuffered from the disease or disorder remains in remission. The termsencompass modulating the threshold, development or duration of thedisease or disorder, or changing the way that a patient responds to thedisease or disorder.

As used herein, and unless otherwise specified, the term “about,” whenused in connection with doses, amounts, or weight percent of ingredientsof a composition or a dosage form, means dose, amount, or weight percentthat is recognized by those of ordinary skill in the art to provide apharmacological effect equivalent to that obtained from the specifieddose, amount, or weight percent is encompassed. Specifically, the term“about” contemplates a dose, amount, or weight percent within 30%, 25%,20%, 15%, 10%, or 5% of the specified dose, amount, or weight percent isencompassed.

As used herein, and unless otherwise specified, the term “stable,” whenused in connection with a formulation or a dosage form, means that theactive ingredient of the formulation or dosage form remains solubilizedfor a specified amount of time and does not significantly degrade oraggregate or become otherwise modified (e.g., as determined, forexample, by HPLC). In some embodiments, about 70% or greater, about 80%or greater or about 90% or greater of the compound remains solubilizedafter the specified period. Stability can also refer to thecompatibility of pharmaceutically acceptable excipients describedherein. Accordingly, a dosage form can be considered stable when thecombined pharmaceutically acceptable excipients and active agent(s)described herein do not degrade or otherwise modify (e.g., react with)the effectiveness or therapeutic value of an active agent describedherein.

As used herein, the term “tumor,” refers to all neoplastic cell growthand proliferation, whether malignant or benign, and all pre-cancerousand cancerous cells and tissues. “Neoplastic,” as used herein, refers toany form of dysregulated or unregulated cell growth, whether malignantor benign, resulting in abnormal tissue growth. Thus, “neoplastic cells”include malignant and benign cells having dysregulated or unregulatedcell growth.

As used herein, “hematologic malignancy” refers to cancer of the body'sblood-forming and immune system—the bone marrow and lymphatic tissue.Such cancers include leukemias, lymphomas (Non-Hodgkin's Lymphoma),Hodgkin's disease (also called Hodgkin's Lymphoma), myelodysplasticsyndrome (MDS), and myeloma. In one embodiment, the myeloma is multiplemyeloma. In some embodiments, the leukemia is, for example, acutemyelogenous leukemia (AML), acute lymphocytic leukemia (ALL), adultT-cell leukemia, chronic lymphocytic leukemia (CLL), hairy cellleukemia, myelodysplasia, myeloproliferative disorders, chronicmyelogenous leukemia (CML), myelodysplastic syndrome (MDS), humanlymphotropic virus-type 1 (HTLV 1) leukemia, mastocytosis, or B-cellacute lymphoblastic leukemia. In some embodiments, the lymphoma is, forexample, diffuse large B-cell lymphoma (DLBCL), B-cell immunoblasticlymphoma, small non-cleaved cell lymphoma, human lymphotropic virus-type1 (HTLV-1) leukemia/lymphoma, adult T-cell lymphoma, peripheral T-celllymphoma (PTCL), cutaneous T-cell lymphoma (CTCL), mantle cell lymphoma(MCL), Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), AIDS-relatedlymphoma, follicular lymphoma, small lymphocytic lymphoma,T-cell/histiocyte rich large B-cell lymphoma, transformed lymphoma,primary mediastinal (thymic) large B-cell lymphoma, splenic marginalzone lymphoma, Richter's transformation, nodal marginal zone lymphoma,or ALK-positive large B-cell lymphoma. In one embodiment, thehematological cancer is indolent lymphoma including, for example, DLBCL,follicular lymphoma, or marginal zone lymphoma.

The term “leukemia” refers to malignant neoplasms of the blood-formingtissues. The leukemia includes, but is not limited to, chroniclymphocytic leukemia, chronic myelocytic leukemia, acute lymphoblasticleukemia, acute myeloid leukemia, and acute myeloblastic leukemia. Theleukemia can be relapsed, refractory or resistant to conventionaltherapy.

The term “myelodysplastic syndrome” refers to hematological conditionscharacterized by abnormalities in the production of one or more of thecellular components of blood (red cells, white cells (other thanlymphocytes) and platelets (or their progenitor cells, megakaryocytes)),and includes the following disorders: refractory anemia (RA); RA withringed sideroblasts (RARS); RA with excess of blasts (RAEB); refractorycytopenia with multilineage dysplasia (RCMD), refractory cytopenia withunilineage dysplasia (RCUD); unclassifiable myelodysplastic syndrome(MDS-U), myelodysplastic syndrome associated with an isolated del(5q)chromosome abnormality, therapy-related myeloid neoplasms and chronicmyelomonocytic leukemia (CMML).

As used herein, “promyelocytic leukemia” or “acute promyelocyticleukemia” refers to a malignancy of the bone marrow in which there is adeficiency of mature blood cells in the myeloid line of cells and anexcess of immature cells called promyelocytes. It is usually marked byan exchange of regions of chromosomes 15 and 17.

As used herein, “acute lymphocytic leukemia (ALL)”, also known as “acutelymphoblastic leukemia” refers to a malignant disease caused by theabnormal growth and development of early nongranular white blood cells,or lymphocytes.

As used herein, “T-cell leukemia” refers to a disease in which certaincells of the lymphoid system called T lymphocytes or T cells aremalignant. T cells are white blood cells that normally can attackvirus-infected cells, foreign cells, and cancer cells and producesubstances that regulate the immune response.

The term “relapsed” refers to a situation where patients who have had aremission of cancer after therapy have a return of cancer cells in themarrow and/or a decrease in normal blood cells.

The term “refractory or resistant” refers to a circumstance wherepatients, even after intensive treatment, have residual cancer cells,for example in their marrow.

As used herein, overall survival (OS) means the time from randomizationin a clinical trial until death from any cause. As used herein,progression-free survival (PFS) means the time from randomization in aclinical trial until progression or death. As used herein, event-freesurvival (EFS) means the time from study entry until any treatmentfailure, including disease progression, treatment discontinuation forany reason, or death. As used herein, overall response rate (ORR) meansthe sum of the percentage of patients who achieve complete and partialresponsess. As used herein, duration of response (DoR) is the time fromachieving a response until relapse or disease progression.

As used herein, the abbreviations for any protective groups, amino acidsand other compounds, are, unless indicated otherwise, in accord withtheir common usage, recognized abbreviations, or the IUPAC-IUBCommission on Biochemical Nomenclature (see, Biochem. 1972, 11:942-944).

6.2 Polymorphs of2-(4-chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamide

In one embodiment, Compound 1 is polymorph Form A, Form B, Form C, FormD, Form E or an amorphous form of2-(4-chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamide.Polymorphs of2-(4-chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamideare described in a U.S. provisional patent application filedconcurrently herewith entitled SOLID FORMS OF2-(4-CHLOROPHENYL)-N-((2-(2,6-DIOXOPIPERIDIN-3-YL)-OXOISOINDOLIN-5-YL)METHYL)-2,2-DIFLUOROACETAMIDE,AND THEIR PHARMACEUTICAL COMPOSITIONS AND USES”, the disclosure of whichis incorporated herein by reference in its entirety. Polymorphs of2-(4-chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamideare briefly described herein.

Form A of2-(4-chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamide

In certain embodiments, the lyophilized formulations provided herein areprepared from Form A of Compound 1.

In one embodiment, Form A is an anhydrous form of Compound 1. In anotherembodiment, Form A of Compound 1 is crystalline.

In certain embodiments, Form A is obtained by crystallization fromcertain solvent systems, for example, solvent systems comprising one ormore of the following solvents: acetone and the solvent mixture ofisopropanol and water at room temperature. In certain embodiments, FormA is obtained as an intermediate solid form from slurries at elevatedtemperature, for example about 50° C., in ethanol/water (1:1), acetoneor acetonitrile.

In certain embodiments, Form A is substantially crystalline, asindicated by, e.g., X-ray powder diffraction measurements. In oneembodiment, Form A of Compound 1 has an X-ray powder diffraction patternsubstantially as shown in FIG. 2.

In one embodiment, Form A of Compound 1 has one or more characteristicX-ray powder diffraction peaks at a two-theta angle of approximately11.5, 15.6, 16.6, 17.2, 18.1, 19.0, 19.6, 21.1, 23.2 or 24.8 degrees 2θas depicted in FIG. 2. In another embodiment, Form A of Compound 1 hasone, two, three or four characteristic X-ray powder diffraction peaks ata two-theta angle of approximately 15.6, 16.6, 17.2 or 24.8 degrees 2θ.In another embodiment, Form A of Compound 1 has one, two, three, four,five, six or seven characteristic X-ray powder diffraction peaks as setforth in Table 1. In another embodiment, Form A of Compound 1 has one,two, or three characteristic X-ray powder diffraction peaks as set forthin Table 1.

In one embodiment, Form A of Compound 1 has the SEM picture as shown inFIG. 3.

In one embodiment, the crystalline form of Compound 1 has athermogravimetric (TGA) thermograph corresponding substantially to therepresentative TGA thermogram as depicted in FIG. 4. In certainembodiments, no TGA weight loss is observed for Form A.

In one embodiment, crystalline form A of Compound 1 has a DSC thermogramcorresponding substantially as depicted in FIG. 5. In certainembodiments, Form A is characterized by a DSC plot comprising a meltingevent with an onset temperature of 229° C. and heat of fusion of 118J/g.

In certain embodiments, Form A is characterized by dynamic vaporsorption analysis. A representative dynamic vapor sorption (DVS)isotherm plot is shown in FIG. 6. In certain embodiments, when therelative humidity (“RH”) is increased from about 0% to about 90% RH,Form A exhibits less than 1.5%, less than 1.2% or about 1.2% w/w wateruptake. In certain embodiments, Form A comprises less than 0.1% water asdetermined in a coulometric Karl Fischer (KF) titrator equipped with anoven sample processor set at 225° C.

In certain embodiments, no significant degradation or residual solventfor Form A is observed by ¹H NMR (FIG. 7).

In certain embodiments, Form A of Compound 1 is characterized by itsstability profile upon compression. In certain embodiments, Form A isstable, e.g., its XRPD pattern remains substantially unchanged withbroader diffraction peaks, upon application of 2000-psi pressure forabout 1 minute (FIG. 8).

In still another embodiment, Form A of Compound 1 is substantially pure.In certain embodiments, the substantially pure Form A of Compound 1 issubstantially free of other solid forms, e.g., amorphous form. Incertain embodiments, the purity of the substantially pure Form A ofCompound 1 is no less than about 95% pure, no less than about 96% pure,no less than about 97% pure, no less than about 98% pure, no less thanabout 98.5% pure, no less than about 99% pure, no less than about 99.5%pure, or no less than about 99.8% pure.

Certain embodiments Form A of Compound 1 is substantially pure. Incertain embodiments herein Form A of Compound 1 is substantially free ofother solid forms comprising Compound 1 including, e.g., Forms B, C, D,E and/or an amorphous solid form comprising Compound 1. In certainembodiments, Form A is a mixture of solid forms comprising Compound 1,including, e.g., a mixture comprising one or more of the following:Forms B, C, D, E and an amorphous solid form comprising Compound 1.

Form B of2-(4-chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamide

In certain embodiments, the lyophilized formulations provided herein areprepared from anhydrous Form B of Compound 1.

In certain embodiments, Form B is obtained by anti-solventrecrystallization from certain solvent systems, for example, solventsystems comprising one or more of the following solvents:methanol/water, DMSO/isopropanol, DMSO/toluene, and DMSO/water. Incertain embodiments, Form B is obtained by cooling recrystallizationfrom THF/water (1:1).

In certain embodiments, Form B is crystalline, as indicated by, e.g.,X-ray powder diffraction measurements. In one embodiment, Form B ofCompound 1 has an X-ray powder diffraction pattern substantially asshown in FIG. 9.

In one embodiment, Form B of Compound 1 has one or more characteristicX-ray powder diffraction peaks at a two-theta angle of approximately15.4, 16.3, 16.7, 17.7, 20.4, 25.6 or 27.5, degrees 2θ as depicted inFIG. 9. In another embodiment, Form B of Compound 1 has one, two, threeor four characteristic X-ray powder diffraction peaks at a two-thetaangle of approximately 16.7, 25.6, 15.4 or 16.3 degrees 2θ. In anotherembodiment, Form B of Compound 1 has one, two, three, four, five, six orseven characteristic X-ray powder diffraction peaks as set forth inTable 2. In another embodiment, Form B of Compound 1 has one, two, orthree characteristic X-ray powder diffraction peaks as set forth inTable 2.

In one embodiment, Form B of Compound 1 has the SEM picture as shown inFIG. 10. In one embodiment, a crystalline form of Compound 1 has athermogravimetric (TGA) thermograph corresponding substantially to therepresentative TGA thermogram as depicted in FIG. 11. In certainembodiments, Form B shows no TGA weight loss below 170° C. In certainembodiments, Form B shows a TGA weight loss of 0.4% between 170˜230° C.

In one embodiment, crystalline Form B of Compound 1 has a DSC thermogramcorresponding substantially as depicted in FIG. 12. In certainembodiments, Form B is characterized by a DSC plot comprising amelt/recrystallization event at 219˜224° C. and a major melting eventwith a peak temperature of 231° C.

In certain embodiments, Form B is characterized by dynamic vaporsorption analysis. A representative dynamic vapor sorption (DVS)isotherm plot is shown in FIG. 13. In certain embodiments, when therelative humidity (“RH”) is increased from about 0% to about 90% RH,Form B exhibits about 1.4% w/w water uptake. In certain embodiments,Form B comprises less than 0.1% water as determined in a coulometricKarl Fischer (KF) titrator equipped with an oven sample processor set at225° C.

In certain embodiments, Form B shows no significant degradation orresidual solvent by ¹H NMR (FIG. 14).

In certain embodiments, Form B of Compound 1 is characterized by itsstability profile upon compression. In certain embodiments, Form B isstable, e.g., its XRPD pattern remains substantially unchanged withbroader diffraction peaks, upon application of 2000-psi pressure forabout 1 minute (FIG. 15).

In still another embodiment, Form B of Compound 1 is substantially pure.In certain embodiments, the substantially pure Form B of Compound 1 issubstantially free of other solid forms, e.g., amorphous form. Incertain embodiments, the purity of the substantially pure Form B ofCompound 1 is no less than about 95% pure, no less than about 96% pure,no less than about 97% pure, no less than about 98% pure, no less thanabout 98.5% pure, no less than about 99% pure, no less than about 99.5%pure, or no less than about 99.8% pure.

Certain embodiments, Form B of Compound 1 is substantially pure. Incertain embodiments, Form B of Compound 1 is substantially free of othersolid forms comprising Compound 1 including, e.g., Forms A, C, D, E,and/or an amorphous solid form comprising Compound 1. In certainembodiments, Form B is a mixture of solid forms comprising Compound 1,including, e.g., a mixture comprising one or more of the following:Forms A, C, D, E, and an amorphous solid form comprising Compound 1.

Form C of2-(4-chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamide

In certain embodiments, the lyophilized formulations provided herein areprepared from anhydrous Form C of Compound 1. In certain embodiments,Form C is the most thermodynamically stable anhydrate among the crystalforms of Compound 1.

In certain embodiments, Form C is obtained by slurrying Compound 1 incertain solvent systems, for example, solvent systems comprising one ormore of the following solvents: acetonitril/water, acetone, orethanol/water for extended period of time.

In certain aspects, Form C is obtained by slurrying Form B (1×wt) inacetone (30×vol) at an elevated temperature, for example, from 60-80° C.or 70-75° C. for at least 24 hours, and cooling the mixture to roomtemperature. In one aspect, the slurrying is conducted at a temperatureof 70-75° C. under nitrogen pressure of 50-55-psi. In one aspect, themixture is cooled to room temperature over at least 6 hours.

In certain embodiments, Form C is crystalline, as indicated by, e.g.,X-ray powder diffraction measurements. In one embodiment, Form C ofCompound 1 has an X-ray powder diffraction pattern substantially asshown in FIG. 16.

In one embodiment, Form C of Compound 1 has one or more characteristicX-ray powder diffraction peaks at a two-theta angle of approximately7.4, 11.5, 15.8, 16.7, 16.9, 17.7, 18.4, 19.2, 19.5, 21.1, 23.4, 24.7,or 29.9, degrees 2θ as depicted in FIG. 16. In another embodiment, FormC of Compound 1 has one, two, three or four characteristic X-ray powderdiffraction peaks at a two-theta angle of approximately 16.7, 16.9, 17.7or 24.7 degrees 2θ. In another embodiment, Form C of Compound 1 has one,two, three, four, five, six or seven characteristic X-ray powderdiffraction peaks as set forth in Table 3. In another embodiment, Form Cof Compound 1 has one, two, or three characteristic X-ray powderdiffraction peaks as set forth in Table 3.

In one embodiment, Form C of Compound 1 has the SEM picture as shown inFIG. 17. In one embodiment, a crystalline form of Compound 1 has athermogravimetric (TGA) thermograph corresponding substantially to therepresentative TGA thermogram as depicted in FIG. 18. In certainembodiments, Form C shows no TGA weight loss.

In one embodiment, crystalline Form C of Compound 1 has a DSC thermogramcorresponding substantially as depicted in FIG. 19. In certainembodiments, Form C is characterized by a DSC plot comprising meltingevent with an onset temperature of 232° C. and heat of fusion of 126J/g.

In certain embodiments, Form C is characterized by dynamic vaporsorption analysis. A representative dynamic vapor sorption (DVS)isotherm plot is shown in FIG. 20. In certain embodiments, when therelative humidity (“RH”) is increased from about 0% to about 90% RH,Form C exhibits about 0.6% w/w water uptake. In certain embodiments,Form C comprises less than 0.1% water as determined in a coulometricKarl Fischer (KF) titrator equipped with an oven sample processor set at225° C.

In certain embodiments, Form C shows no significant degradation orresidual solvent by ¹H NMR (FIG. 21).

In certain embodiments, Form C of Compound 1 is characterized by itsstability profile upon compression. In certain embodiments, Form C isstable, e.g., its XRPD pattern remains substantially unchanged withbroader diffraction peaks, upon application of 2000-psi pressure forabout 1 minute (FIG. 22).

In still another embodiment, Form C of Compound 1 is substantially pure.In certain embodiments, the substantially pure Form C of Compound 1 issubstantially free of other solid forms, e.g., amorphous form. Incertain embodiments, the purity of the substantially pure Form C ofCompound 1 is no less than about 95% pure, no less than about 96% pure,no less than about 97% pure, no less than about 98% pure, no less thanabout 98.5% pure, no less than about 99% pure, no less than about 99.5%pure, or no less than about 99.8% pure.

In certain embodiments, Form C of Compound 1 is substantially pure. Incertain embodiments, Form C of Compound 1 is substantially free of othersolid forms comprising Compound 1 including, e.g., Forms A, B, D, E,and/or an amorphous solid form comprising Compound 1. In certainembodiments, Form C is a mixture of solid forms comprising Compound 1,including, e.g., a mixture comprising one or more of the following:Forms A, B, D, E, and an amorphous solid form comprising Compound 1.

Form D of2-(4-chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamide

In certain embodiments, the lyophilized formulations provided herein areprepared from Form D of Compound 1. In certain embodiments, Form D ofCompound 1 is a DMSO solvate.

In certain embodiments, Form D is obtained by heating Form B inDMSO/methyl isobutyl ketone and cooling the solution.

In certain embodiments, Form D is crystalline, as indicated by, e.g.,X-ray powder diffraction measurements. In one embodiment, Form D ofCompound 1 has an X-ray powder diffraction pattern substantially asshown in FIG. 23.

In one embodiment, Form D of Compound 1 has one or more characteristicX-ray powder diffraction peaks at a two-theta angle of approximately14.1, 14.3, 18.8, 19.1, 23.6 or 24.0 degrees 2θ as depicted in FIG. 23.In another embodiment, Form D of Compound 1 has one, two, three or fourcharacteristic X-ray powder diffraction peaks at a two-theta angle ofapproximately 14.1, 14.3, 18.8 or 19.1 degrees 2θ. In anotherembodiment, Form D of Compound 1 has one, two, three, four, five, six orseven characteristic X-ray powder diffraction peaks as set forth inTable 4. In another embodiment, Form D of Compound 1 has one, two, orthree characteristic X-ray powder diffraction peaks as set forth inTable 4.

In one embodiment, provided herein is a crystalline form of Compound 1having a thermogravimetric (TGA) thermograph corresponding substantiallyto the representative TGA thermogram as depicted in FIG. 24. In certainembodiments, Form D shows TGA weight loss of about 14.1% up to 140° C.

In certain embodiments, Form D comprises DMSO in about 14.3 wt % asmeasured by gas chromatography.

In still another embodiment, Form D of Compound 1 is substantially pure.In certain embodiments, the substantially pure Form D of Compound 1 issubstantially free of other solid forms, e.g., amorphous form. Incertain embodiments, the purity of the substantially pure Form D ofCompound 1 is no less than about 95% pure, no less than about 96% pure,no less than about 97% pure, no less than about 98% pure, no less thanabout 98.5% pure, no less than about 99% pure, no less than about 99.5%pure, or no less than about 99.8% pure.

In certain embodiments Form D of Compound 1 is substantially pure. Incertain embodiments, Form D of Compound 1 is substantially free of othersolid forms comprising Compound 1 including, e.g., Forms A, B, C, E,and/or an amorphous solid form comprising Compound 1 as provided herein.In certain embodiments, Form D is a mixture of solid forms comprisingCompound 1, including, e.g., a mixture comprising one or more of thefollowing: Forms A, B, C, E, and an amorphous solid form comprisingCompound 1.

Form E of2-(4-chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamide

In certain embodiments, the lyophilized formulations provided herein areprepared from Form E of Compound 1. In certain embodiments, Form E ofCompound 1 is a DMSO solvate.

In certain embodiments, Form E is obtained from Form C in DMSO/MIBK orDMSO/IPA or DMSO/anisole at room temperature.

In certain embodiments, Form E is crystalline, as indicated by, e.g.,X-ray powder diffraction measurements. In one embodiment, Form E ofCompound 1 has an X-ray powder diffraction pattern substantially asshown in FIG. 25.

In one embodiment, Form E of Compound 1 has one or more characteristicX-ray powder diffraction peaks at a two-theta angle of approximately10.5, 12.5, 16.1, 17.0, 18.5, 21.2, 21.7, 22.6, 22.9, 23.4, 23.8, 24.1,25.1 or 26.7, degrees 2θ as depicted in FIG. 25. In another embodiment,Form E of Compound 1 has one, two, three or four characteristic X-raypowder diffraction peaks at a two-theta angle of approximately 16.1,17.0, 21.2 or 22.9 degrees 2θ. In another embodiment, Form E of Compound1 has one, two, three, four, five, six or seven characteristic X-raypowder diffraction peaks as set forth in Table 5. In another embodiment,Form E of Compound 1 has one, two, or three characteristic X-ray powderdiffraction peaks as set forth in Table 5.

In one embodiment, provided herein is a crystalline form of Compound 1having a thermogravimetric (TGA) thermograph corresponding substantiallyto the representative TGA thermogram as depicted in FIG. 26. In certainembodiments, Form E shows TGA weight loss of about 19.4% up to 120° C.In certain embodiments, Form E shows additional weight loss of 24.9%between 120 and 220° C.

In one embodiment, Form E of Compound 1 is substantially pure. Incertain embodiments, the substantially pure Form E of Compound 1 issubstantially free of other solid forms, e.g., amorphous form. Incertain embodiments, the purity of the substantially pure Form E ofCompound 1 is no less than about 95% pure, no less than about 96% pure,no less than about 97% pure, no less than about 98% pure, no less thanabout 98.5% pure, no less than about 99% pure, no less than about 99.5%pure, or no less than about 99.8% pure.

In certain embodiments, Form E of Compound 1 is substantially pure. Incertain embodiments herein, Form E of Compound 1 is substantially freeof other solid forms comprising Compound 1 including, e.g., Forms A, B,C, D and/or an amorphous solid form comprising Compound 1. In certainembodiments, Form E is a mixture of solid forms comprising Compound 1,including, e.g., a mixture comprising one or more of the following:Forms A, B, C, D and an amorphous solid form comprising Compound 1.

Amorphous Form of2-(4-chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamide

In certain embodiments, the lyophilized formulations provided hereincomprise amorphous Compound 1.

In certain embodiments, provided herein are methods for making theamorphous form by heating Compound 1 in THF and water and cooling thesolution.

In one embodiment, provided herein is an amorphous solid form ofCompound 1 having a modulated DSC thermogram as depicted in FIG. 27.

In one embodiment, amorphous Compound 1 has an X-ray powder diffractionpattern substantially as shown in FIG. 28.

In one embodiment, amorphous Compound 1 has a ¹H NMR spectrumsubstantially as shown in FIG. 29.

In still another embodiment, amorphous Compound 1 is substantially pure.In certain embodiments, the substantially pure amorphous Compound 1 issubstantially free of other solid forms, e.g., Form A, Form B, Form C,Form D or Form E. In certain embodiments, the purity of thesubstantially pure amorphous Compound 1 is no less than about 95% pure,no less than about 96% pure, no less than about 97% pure, no less thanabout 98% pure, no less than about 98.5% pure, no less than about 99%pure, no less than about 99.5% pure, or no less than about 99.8% pure.

6.3 Exemplary Formulations

Provided herein are stable lyophilized formulations of Compound 1. Inone embodiment, the lyophilized formulations of Compound 1 comprise asolid form of2-(4-chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamide.In one embodiment, the lyophilized formulations of Compound 1 comprisean amorphous form of2-(4-chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamide.

In certain embodiment, the lyophilized formulations provided hereincomprise Compound 1, a buffer and a bulking agent. In one embodiment, alyophilized formulation provided herein comprises about 0.1-2% Compound1, about 2-15% buffer and about 70-95% bulking agent based on the totalweight of the lyophilized formulation.

In one aspect, the lyophilized formulation provided herein comprisesCompound 1 in an amount of about 0.1 to about 2% based on the totalweight of the lyophilized formulation. In certain embodiments, theamount of Compound 1 is from about 0.1% to about 1.5%, about 0.1% toabout 1% or about 0.35% to about 0.9% based on the total weight of thelyophilized formulation. In certain embodiments, the amount of Compound1 is about 0.1%, 0.2%, 0.3%, 0.35%, 0.36%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%,0.9%, or 1.0% based on the total weight of the lyophilized formulation.In one embodiment, the amount of Compound 1 in the lyophilizedformulation is about 0.3 to about 0.4% based on the total weight of thelyophilized formulation. In one embodiment, the amount of Compound 1 inthe lyophilized formulation is about 0.36% based on the total weight ofthe lyophilized formulation. In one embodiment, the amount of Compound 1in the lyophilized formulation is about 0.9 to about 1% based on thetotal weight of the lyophilized formulation. In one embodiment, theamount of Compound 1 in the lyophilized formulation is about 0.93% basedon the total weight of the lyophilized formulation.

In another aspect is a lyophilized formulation that comprises Compound 1in an amount of about 0.1 mg to about 5 mg in a 20 cc vial. In stillanother aspect is a lyophilized formulation that comprises Compound 1 inan amount of about 0.1 mg to about 5 mg, about 0.1 mg to about 4 mg,about 0.1 mg to about 3 mg, about 0.1 mg to about 2 mg, about 0.5 mg toabout 5 mg, about 0.5 mg to about 3 mg, about 0.5 mg to about 2 mg,about 0.5 mg to about 1.5 mg in a 20 cc vial. In one aspect Compound 1is present in an amount of about 0.5, 0.6, 0.7, 0.75, 0.76, 0.8, 0.9,1.0, 1.2 mg in a 20 cc vial. In one aspect Compound 1 is present in anamount of about 0.76 mg in a 20 cc vial. In one aspect Compound 1 ispresent in an amount of about 1 mg in a 20 cc vial.

In one aspect, the lyophilized formulations provided herein contain acitrate buffer. In one aspect, the amount of citrate buffer in theformulations provided herein is from about 5% to about 25% based ontotal weight of the lyophilized formulation. In one aspect, the amountof citrate buffer in the formulations provided herein is about 10, 11,12, 12.5, 12.7, 12.78, 12.8, 13, 14, 15, 16, 17, 17.3, 17.42, 17.5,17.7, 18, 19 or 20% based on total weight of the lyophilizedformulation. In one aspect, the amount of citrate buffer in theformulations provided herein is about 12.78% based on total weight ofthe lyophilized formulation. In one aspect, the amount of citrate bufferin the formulations provided herein is about 17.42% based on totalweight of the lyophilized formulation.

In one embodiment, the citrate buffer comprises anhydrous citric acidand anhydrous sodium citrate. In certain embodiments, the amount ofanhydrous citric acid is from about 2% to about 10%, about 3% to about9%, about 5% to about 8% or about 6% to about 8% based on total weightof the lyophilized formulation. In certain embodiments, the amount ofanhydrous citric acid in the lyophilized formulation is about 2%, 4%,6%, 6.2%, 6.4%, 6.6%, 6.8%, 7%, 7.3%, 7.4%, 7.5%, 8%, 8.5% or 9% basedon total weight of the lyophilized formulation. In one embodiment, theamount of anhydrous citric acid in the lyophilized formulation is about6%, 6.2%, 6.4%, 6.41%, 6.6%, 6.8% or 7% based on total weight of thelyophilized formulation. In one embodiment, the amount of anhydrouscitric acid in the lyophilized formulation is about 7%, 7.3%, 7.4%,7.43%, 7.5% or 8% based on total weight of the lyophilized formulation.In one embodiment, the amount of anhydrous citric acid in thelyophilized formulation is about 6.41% based on total weight of thelyophilized formulation. In one embodiment, the amount of anhydrouscitric acid in the lyophilized formulation is about 7.43% based on totalweight of the lyophilized formulation.

In still another aspect is a lyophilized formulation that comprisesanhydrous citric acid in an amount of about 5 mg to about 20 mg in a 20cc vial. In one embodiment, the amount of anhydrous citric acid is about5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mg in a 20cc vial. In one embodiment, the amount of anhydrous citric acid is about17.7 mg in a 20 cc vial. In one embodiment, the amount of anhydrouscitric acid is about 6.1 mg in a 20 cc vial.

In certain embodiments, the amount of anhydrous sodium citrate is fromabout 2% to about 15%, about 4% to about 15% or about 5% to about 10%based on total weight of the lyophilized formulation. In certainembodiments, the amount of anhydrous sodium citrate in the lyophilizedformulation is about 2%, 3%, 4%, 5%, 6%, 6.2%, 6.37%, 6.4%, 6.6%, 6.8%,7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 12% or about 15% based on totalweight of the lyophilized formulation. In one embodiment, the amount ofanhydrous sodium citrate in the lyophilized formulation is about 6%,6.2%, 6.37% 6.4%, 6.6%, 6.8% or 7% based on total weight of thelyophilized formulation. In one embodiment, the amount of anhydroussodium citrate in the lyophilized formulation is about 8%, 8.5%, 9%,9.5%, 9.99%, 10% or 10.5% based on total weight of the lyophilizedformulation. In one embodiment, the amount of anhydrous sodium citratein the lyophilized formulation is about 6.37% based on total weight ofthe lyophilized formulation. In one embodiment, the amount of anhydroussodium citrate in the lyophilized formulation is about 9.99% based ontotal weight of the lyophilized formulation.

In still another aspect is a lyophilized formulation that comprisesanhydrous sodium citrate in an amount of about 5 mg to about 20 mg in a20 cc vial. In one embodiment, the amount of anhydrous sodium citrate isabout 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mg ina 20 cc vial. In one embodiment, the amount of anhydrous sodium citrateis about 17.6 mg in a 20 cc vial. In one embodiment, the amount ofanhydrous sodium citrate is about 8.2 mg in a 20 cc vial.

In certain embodiments, the amount of anhydrous citric acid in thelyophilized formulation is about 2%, 4%, 6%, 6.2%, 6.4%, 6.6%, 6.8%, 7%,7.3%, 7.4%, 7.5%, 8%, 8.5% or 9% and the amount of anhydrous sodiumcitrate in the lyophilized formulation is about 2%, 3%, 4%, 5%, 6%,6.2%, 6.4%, 6.6%, 6.8%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 12% or about15% based on total weight of the lyophilized formulation. In oneembodiment, the amount of anhydrous citric acid in the lyophilizedformulation is about 6%, 6.2%, 6.4%, 6.6%, 6.8% or 7% and the amount ofanhydrous sodium citrate in the lyophilized formulation is about 6%,6.2%, 6.4%, 6.6%, 6.8% or 7% based on total weight of the lyophilizedformulation. In one embodiment, the amount of anhydrous citric acid inthe lyophilized formulation is about 7%, 7.3%, 7.4%, 7.5% or 8% and theamount of anhydrous sodium citrate in the lyophilized formulation isabout 8%, 8.5%, 9%, 9.5%, 10% or 10.5% based on total weight of thelyophilized formulation. In one embodiment, the amount of anhydrouscitric acid is about 6.1 mg and the amount of anhydrous sodium citrateis about 8.2 mg in a 20 cc vial. In one embodiment, the amount ofanhydrous citric acid is about 17.7 mg and the amount of anhydroussodium citrate is about 17.6 mg in a 20 cc vial.

In one aspect, the bulking agent in the lyophilized formulationsprovided herein comprises Captisol®, mannitol or Kleptose®, for example,β-cyclodextrin, hydroxypropyl β-cyclodextrin and methylatedβ-cyclodextrin. In certain embodiments, the bulking agent in thelyophilized formulations provided herein comprises Kleptose®hydroxypropyl β-cyclodextrins (Kleptose®HPB). In certain embodiments,the amount of the bulking agent in the lyophilized compositions providedherein is from about 70% to about 95%, about 75% to about 90% or about80% to about 90% based on total weight of the lyophilized formulation.In certain embodiments, the amount of hydroxypropyl β-cyclodextrin inthe lyophilized compositions provided herein is from about 70% to about95%, about 75% to about 90% or about 80% to about 90% based on totalweight of the lyophilized formulation. In certain embodiments, theamount of hydroxypropyl β-cyclodextrin in the lyophilized compositionsprovided herein is about 75%, 80%, 81%, 81.61%, 82%, 83%, 84%, 85%, 86%,86.86%, 87%, 88%, 89% or 90% based on total weight of the lyophilizedformulation. In one embodiment, the amount of hydroxypropylβ-cyclodextrin in the lyophilized compositions provided herein is about86.86% based on total weight of the lyophilized formulation. In oneembodiment, the amount of hydroxypropyl β-cyclodextrin in thelyophilized compositions provided herein is about 81.61% based on totalweight of the lyophilized formulation.

In another aspect is a lyophilized formulation that comprisesKleptose®HPB in an amount of about 67 mg in a 20 cc vial. In stillanother aspect is a lyophilized formulation that comprises Kleptose®HPBin an amount of about 240 mg in a 20 cc vial.

In certain embodiments, the lyophilized formulation upon reconstitutionhas a pH of about 4 to 5. In one embodiment, the lyophilized formulationupon reconstitution has a pH of about 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6,4.7, 4.8, 4.9 or 5.

In certain embodiments, provided herein is a container comprising alyophilized composition provided herein. In one aspect, the container isa glass vial. In one aspect, the container is a 20 cc glass vial.

In certain embodiments, the lyophilized formulation has a composition asdescribed in the Table 21. In certain embodiments, the lyophilizedformulation has a composition as described in the Table 34.

The lyophilized formulations of Compound 1 provided herein can beadministered to a patient in need thereof using standard therapeuticmethods for delivering Compound 1 including, but not limited to, themethods described herein. In one embodiment, the lyophilizedformulations provided herein are reconstituted in a pharmaceuticallyacceptable solvent to produce a pharmaceutically acceptable solution,wherein the solution is administered (such as by intravenous injection)to the patient.

The lyophilized formulation provided herein can be constituted forparenteral administration to a patient using any pharmaceuticallyacceptable diluent. Such diluents include, but are not limited toSterile Water for Injection (SWFI), Dextrose 5% in Water (D5W), or acosolvent system. Any quantity of diluent may be used to constitute thelyophilized formulation such that a suitable solution for injection isprepared. Accordingly, the quantity of the diluent must be sufficient todissolve the lyophilized formulation. In one embodiment, 1-5 mL or 1 to3 mL of a diluent are used to constitute the lyophilized formulation toyield a final concentration of, about 0.1-5 mg/mL, about 0.1-1 mg/mL,about 0.5-1 mg/mL of Compound 1. In certain embodiments, the finalconcentration of Compound 1 in the reconstituted solution is about 0.5mg/mL. In certain embodiment, the volume of the reconstitution diluentvaries between 2 ml and 20 ml to yield a final concentration of 0.05-0.5mg/mL. In certain embodiment, depending on the required dose, multiplevials may be used for reconstitution.

The constituted solutions of lyophilized formulation can be stored andused within up to about 24 hours, about 12 hours or about 8 hours. Insome embodiments, the solution is used within 8 hour of preparation. Insome embodiments, the solution is used within 5 hour of preparation. Insome embodiments, the solution is used within 1 hour of preparation.

The lyophilized formulation can be a formulation as set forth in Table21 and/or Table 34. Thus, in certain embodiments, the lyophilizedformulation is represented by the designations in Table 21 and/or Table34 (e.g., Formulation IA, Formulation IC, Formulation II, FormulationIII, Formulation IX, or Formulation ID). In one embodiment, theformulation is Formulation IX. In one embodiment, the formulation isFormulation IC. In one embodiment, the formulation is Formulation ID.

In one aspect provided herein is a lyophilized formulation in a 20 ccvial that includes: Compound 1 at an amount that provides 1 mg2-(4-chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamideand a pharmaceutically acceptable carrier or excipient that includes abuffer and bulking agent as described herein. The buffer and bulkingagent can be present at an amount as described herein.

In one aspect provided herein is a lyophilized formulation in a 20 ccvial that includes: Compound 1 at an amount that provides 1 mg2-(4-chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamide,17.7 mg anhydrous citric acid, 17.6 mg anhydrous sodium citrate and 240mg HPB as described herein. In one embodiment, the lyophilizedformulation in a 20 cc vial is reconstituted with 2 mL sterile water forinjection.

In one aspect provided herein is an aqueous composition comprising alyophilized formulation provided herein. In one embodiment, the aqueoussolution comprises 0.5 mg/mL Compound 1.

6.4 Process for Making Formulations

The lyophilized formulations provided herein can be prepared by any ofthe methods known in the art and as described herein, but all methodsinclude the step of bringing the active ingredient into association withthe pharmaceutically acceptable excipient, which constitutes one or morenecessary ingredients (such as buffer and bulking agent).

In one aspect, the formulations provided herein are prepared bydissolving Compound 1 and a bulking agent in a citrate buffer to obtaina solution and lyophilizing the solution. A flow chart illustrating anexemplary process is provided in FIG. 32. In one embodiment, the processcomprises dissolving Kleptose® HPB in a 20 mM, pH 4.3 citrate buffer toobtain a mixture, adding Compound 1 dissolved in DMA to the mixture toobtain a solution, filtering the solution into a 20 cc vial, andlyophilizing the solution. In one embodiment, the solution is filteredthrough one or more 0.45 μm and/or 0.22 μm filters. In one embodiment,the vial is sealed under nitrogen after lyophilization.

In one aspect, the lyophilization process contains three stages:freezing, primary drying, and secondary drying. A liquid formulation istransformed to a lyophilized powder form by going through completesolidification through freezing stage, sublimation of ice and solventsthrough primary drying, and desorption of residual moisture and solventsthrough secondary drying. The shelf temperature and chamber pressure inthe primary drying and secondary drying are controlled to obtained thedesired quality of the finished drug product. In one aspect of theprocess, the cake appearance and structure was characterized by visualinspection.

6.5 Kits

Pharmaceutical packs or kits which comprise pharmaceutical compositionsor dosage forms provided herein are also provided. Exemplary kitsinclude notice in the form prescribed by a governmental agencyregulating the manufacture, use or sale of pharmaceuticals products,which notice reflects approval by the agency of manufacture, use or salefor human administration

6.6 Methods of Treating

In one embodiment, provided herein is a method of treating andpreventing cancer, which comprises administering to a patient alyophilized formulation of Compound 1 provided herein.

In another embodiment, provided herein is method of managing cancer,which comprises administering to a patient a lyophilized formulation ofCompound 1 provided herein.

Also provided herein are methods of treating patients who have beenpreviously treated for cancer but are non-responsive to standardtherapies, as well as those who have not previously been treated. Theinvention also encompasses methods of treating patients regardless ofpatient's age, although some diseases or disorders are more common incertain age groups. The invention further encompasses methods oftreating patients who have undergone surgery in an attempt to treat thedisease or condition at issue, as well as those who have not. Becausepatients with cancer have heterogeneous clinical manifestations andvarying clinical outcomes, the treatment given to a patient may vary,depending on his/her prognosis. The skilled clinician will be able toreadily determine without undue experimentation specific secondaryagents, types of surgery, and types of non-drug based standard therapythat can be effectively used to treat an individual patient with cancer.

As used herein, the term “cancer” includes, but is not limited to, solidtumors and blood borne tumors. The term “cancer” refers to disease ofskin tissues, organs, blood, and vessels, including, but not limited to,cancers of the bladder, bone, blood, brain, breast, cervix, chest,colon, endrometrium, esophagus, eye, head, kidney, liver, lymph nodes,lung, mouth, neck, ovaries, pancreas, prostate, rectum, stomach, testis,throat, and uterus. Specific cancers include, but are not limited to,advanced malignancy, amyloidosis, neuroblastoma, meningioma,hemangiopericytoma, multiple brain metastase, glioblastoma multiforms,glioblastoma, brain stem glioma, poor prognosis malignant brain tumor,malignant glioma, recurrent malignant glioma, anaplastic astrocytoma,anaplastic oligodendroglioma, neuroendocrine tumor, rectaladenocarcinoma, colorectal cancer, including stage 3 and stage 4,unresectable colorectal carcinoma, metastatic hepatocellular carcinoma,Kaposi's sarcoma, karotype acute myeloblastic leukemia, Hodgkin'slymphoma, non-Hodgkin's lymphoma, cutaneous T-Cell lymphoma, cutaneousB-Cell lymphoma, diffuse large B-Cell lymphoma, low grade follicularlymphoma, malignant melanoma, malignant mesothelioma, malignant pleuraleffusion mesothelioma syndrome, peritoneal carcinoma, papillary serouscarcinoma, gynecologic sarcoma, soft tissue sarcoma, scleroderma,cutaneous vasculitis, Langerhans cell histiocytosis, leiomyosarcoma,fibrodysplasia ossificans progressive, hormone refractory prostatecancer, resected high-risk soft tissue sarcoma, unrescectablehepatocellular carcinoma, Waldenstrom's macroglobulinemia, smolderingmyeloma, indolent myeloma, fallopian tube cancer, androgen independentprostate cancer, androgen dependent stage IV non-metastatic prostatecancer, hormone-insensitive prostate cancer, chemotherapy-insensitiveprostate cancer, papillary thyroid carcinoma, follicular thyroidcarcinoma, medullary thyroid carcinoma, and leiomyoma.

In certain embodiments, the cancer is a solid tumor. In certainembodiments, the solid tumor is metastatic. In certain embodiments, thesolid tumor is drug-resistant. In certain embodiments, the solid tumoris hepatocellular carcinoma, prostate cancer, ovarian cancer, orglioblastoma.

In certain embodiments, the cancer is a blood borne tumor. In certainembodiments, the blood borne tumor is metastatic. In certainembodiments, the blood borne tumor is drug resistant. In certainembodiments, the cancer is leukemia. In another embodiment, the canceris MDS.

In one embodiment, methods provided herein encompass treating,preventing and/or managing various types of leukemias such as chroniclymphocytic leukemia (CLL), chronic myelocytic leukemia (CML), acutelymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and acutemyeloblastic leukemia (AML) by administering a therapeutically effectiveamount of a lyophilized formulation provided herein.

In some embodiments, the methods provided herein encompass treating,preventing and/or managing acute leukemia in a subject. In someembodiments, the acute leukemia is acute myeloid leukemia (AML), whichincludes, but is not limited to, undifferentiated AML (M0), myeloblasticleukemia (M1), myeloblastic leukemia (M2), promyelocytic leukemia (M3 orM3 variant (M3V)), myelomonocytic leukemia (M4 or M4 variant witheosinophilia (M4E)), monocytic leukemia (M5), erythroleukemia (M6), andmegakaryoblastic leukemia (M7). In one embodiment, the acute myeloidleukemia is undifferentiated AML (M0). In one embodiment, the acutemyeloid leukemia is myeloblastic leukemia (M1). In one embodiment, theacute myeloid leukemia is myeloblastic leukemia (M2). In one embodiment,the acute myeloid leukemia is promyelocytic leukemia (M3 or M3 variant(M3V)). In one embodiment, the acute myeloid leukemia is myelomonocyticleukemia (M4 or M4 variant with eosinophilia (M4E)). In one embodiment,the acute myeloid leukemia is monocytic leukemia (M5). In oneembodiment, the acute myeloid leukemia is erythroleukemia (M6). In oneembodiment, the acute myeloid leukemia is megakaryoblastic leukemia(M7).

In certain embodiments, the methods of treating, preventing and/ormanaging acute myeloid leukemia in a subject comprise the step ofadministering to the subject an amount of a lyophilized formulation ofCompound 1 provided herein effective to treat, prevent and/or manageacute myeloid leukemia alone or in combination.

In one embodiment, provided herein are methods of treating, preventingand/or managing acute myeloid leukemia by intravenous administration ofa lyophilized formulation of Compound 1. In one embodiment, the alyophilized formulation of Compound 1 is dissolved in water to form anaqueous solution for intravenous administration in methods of treating,preventing and/or managing acute myeloid leukemia provided herein.

In some embodiments, the methods comprise the step of administering tothe subject a lyophilized formulation of Compound 1 provided herein incombination with a second active agent in amounts effective to treat,prevent and/or manage acute myeloid leukemia.

In some embodiments, the methods provided herein encompass treating,preventing and/or managing acute lymphocytic leukemia (ALL) in asubject. In some embodiments, acute lymphocytic leukemia includesleukemia that originates in the blast cells of the bone marrow(B-cells), thymus (T-cells), and lymph nodes. The acute lymphocyticleukemia can be categorized according to the French-American-British(FAB) Morphological Classification Scheme as L1—Mature-appearinglymphoblasts (T-cells or pre-B-cells), L2—Immature and pleomorphic(variously shaped) lymphoblasts (T-cells or pre-B-cells), andL3—Lymphoblasts (B-cells; Burkitt's cells). In one embodiment, the acutelymphocytic leukemia originates in the blast cells of the bone marrow(B-cells). In one embodiment, the acute lymphocytic leukemia originatesin the thymus (T-cells). In one embodiment, the acute lymphocyticleukemia originates in the lymph nodes. In one embodiment, the acutelymphocytic leukemia is L1 type characterized by mature-appearinglymphoblasts (T-cells or pre-B-cells). In one embodiment, the acutelymphocytic leukemia is L2 type characterized by immature andpleomorphic (variously shaped) lymphoblasts (T-cells or pre-B-cells). Inone embodiment, the acute lymphocytic leukemia is L3 type characterizedby lymphoblasts (B-cells; Burkitt's cells). In certain embodiments, theacute lymphocytic leukemia is T-cell leukemia. In one embodiment, theT-cell leukemia is peripheral T-cell leukemia. In another embodiment,the T-cell leukemia is T-cell lymphoblastic leukemia. In anotherembodiment, the T-cell leukemia is cutaneous T-cell leukemia. In anotherembodiment, the T-cell leukemia is adult T-cell leukemia. Thus, themethods of treating, preventing and/or managing acute lymphocyticleukemia in a subject comprise the step of administering to the subjectan amount of a lyophilized formulation of Compound 1 provided hereineffective to treat, prevent and/or manage acute lymphocytic leukemiaalone or in combination with a second active agent. In some embodiments,the methods comprise the step of administering to the subject alyophilized formulation of Compound 1 provided herein in combinationwith a second active agent in amounts effective to treat, prevent and/ormanage acute lymphocytic leukemia.

In some embodiments, the methods provided herein encompass treating,preventing and/or managing chronic myelogenous leukemia (CML) in asubject. The methods comprise the step of administering to the subjectan amount of a lyophilized formulation of Compound 1 provided hereineffective to treat, prevent and/or manage chronic myelogenous leukemia.In some embodiments, the methods comprise the step of administering tothe subject a lyophilized formulation of Compound 1 provided herein incombination with a second active agent in amounts effective to treat,prevent and/or manage chronic myelogenous leukemia.

In some embodiments, the methods provided herein encompass treating,preventing and/or managing chronic lymphocytic leukemia (CLL) in asubject. The methods comprise the step of administering to the subjectan amount of a lyophilized formulation of Compound 1 provided hereineffective to treat, prevent and/or manage chronic lymphocytic leukemia.In some embodiments, the methods comprise the step of administering tothe subject a lyophilized formulation of Compound 1 provided herein incombination with a second active agent in amounts effective to treat,prevent and/or manage chronic lymphocytic leukemia.

In certain embodiments, provided herein are methods of treating,preventing, and/or managing disease in patients with impaired renalfunction. In certain embodiments, provided herein are method oftreating, preventing, and/or managing cancer in patients with impairedrenal function. In certain embodiments, provided herein are methods ofproviding appropriate dose adjustments for patients with impaired renalfunction due to, but not limited to, disease, aging, or other patientfactors.

In certain embodiments, provided herein are methods of treating,preventing, and/or managing lymphoma, including non-Hodgkin's lymphoma.In some embodiments, provided herein are methods for the treatmentand/or management of non-Hodgkin's lymphoma (NHL), including but notlimited to, diffuse large B-cell lymphoma (DLBCL), using prognosticfactors.

In certain embodiments, provided herein are methods of treating,preventing, and/or managing multiple myeloma, includingrelapsed/refractory multiple myeloma in patients with impaired renalfunction or a symptom thereof, comprising administering atherapeutically effective amount of a lyophilized formulation ofCompound 1 provided herein to a patient having relapsed/refractorymultiple myeloma with impaired renal function.

In one embodiment, provided herein are methods of treating, preventing,and/or managing a myelodysplastic syndrome (MDS) in a subject. Themethods comprise the step of administering to the subject atherapeutically active amount of a lyophilized formulation of Compound 1provided herein. In one embodiment, the MDS is relapsed, resistant orrefractory MDS. In one embodiment, MDS is selected from refractoryanemia (RA); RA with ringed sideroblasts (RARS); RA with excess ofblasts (RAEB); refractory cytopenia with multilineage dysplasia (RCMD),refractory cytopenia with unilineage dysplasia (RCUD); unclassifiablemyelodysplastic syndrome (MDS-U), myelodysplastic syndrome associatedwith an isolated del(5q) chromosome abnormality, therapy-related myeloidneoplasms and chronic myelomonocytic leukemia (CMML). In someembodiments, the methods comprise the step of administering to thesubject a lyophilized formulation of Compound 1 provided herein incombination with a second active agent in amounts effective to treat,prevent and/or manage MDS.

In certain embodiments, a therapeutically or prophylactically effectiveamount of Compound 1 is from about 0.005 to about 1,000 mg per day, fromabout 0.01 to about 500 mg per day, from about 0.01 to about 250 mg perday, from about 0.01 to about 100 mg per day, from about 0.1 to about100 mg per day, from about 0.5 to about 100 mg per day, from about 1 toabout 100 mg per day, from about 0.01 to about 50 mg per day, from about0.1 to about 50 mg per day, from about 0.5 to about 50 mg per day, fromabout 1 to about 50 mg per day, from about 0.02 to about 25 mg per day,from about 0.05 to about 10 mg per day, from about 0.05 to about 5 mgper day, from about 0.1 to about 5 mg per day, or from about 0.5 toabout 5 mg per day.

In certain embodiments, the therapeutically or prophylacticallyeffective amount is about 0.1, about 0.2, about 0.5, about 1, about 2,about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10,about 15, about 20, about 25, about 30, about 40, about 45, about 50,about 60, about 70, about 80, about 90, about 100, or about 150 mg perday. In some such embodiments, the therapeutically or prophylacticallyeffective amount is about 2, about 3, about 4, about 5, about 6 or about7 mg per day.

In one embodiment, the recommended daily dose range of Compound 1, forthe conditions described herein lie within the range of from about 0.05mg to about 50 mg per day, preferably given as a single once-a-day dose,or in divided doses throughout a day. In some embodiments, the dosageranges from about 1 mg to about 50 mg per day. In other embodiments, thedosage ranges from about 0.5 to about 5 mg per day. Specific doses perday include 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49or 50 mg per day.

In a specific embodiment, the recommended starting dosage may be 0.1,0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25 or 50 mg per day. In anotherembodiment, the recommended starting dosage may be 0.1, 0.5, 1, 2, 3, 4,or 5 mg per day. The dose may be escalated to 15, 20, 25, 30, 35, 40, 45and 50 mg/day. In a specific embodiment, Compound 1 can be administeredin an amount of about 25 mg/day to patients with leukemia, includingAML. In a particular embodiment, Compound 1 can be administered in anamount of about 10 mg/day to patients with leukemia, including AML. In aparticular embodiment, Compound 1 can be administered in an amount ofabout 5 mg/day to patients with leukemia, including AML. In a particularembodiment, Compound 1 can be administered in an amount of about 4mg/day to patients with leukemia, including AML. In a particularembodiment, Compound 1 provided herein can be administered in an amountof about 3 mg/day to patients with leukemia, including AML. In aparticular embodiment, Compound 1 provided herein can be administered inan amount of about 2 mg/day to patients with leukemia, including AML. Ina particular embodiment, Compound 1 provided herein can be administeredin an amount of about 1 mg/day to patients with leukemia, including AML.In a particular embodiment, Compound 1 provided herein can beadministered in an amount of about 0.5 mg/day to patients with leukemia,including AML.

In a specific embodiment, Compound 1 can be administered in an amount ofabout 25 mg/day to patients with MDS. In a particular embodiment,Compound 1 can be administered in an amount of about 10 mg/day topatients with MDS. In a particular embodiment, Compound 1 can beadministered in an amount of about 5 mg/day to patients with MDS. In aparticular embodiment, Compound 1 can be administered in an amount ofabout 4 mg/day to patients with MDS. In a particular embodiment,Compound 1 provided herein can be administered in an amount of about 3mg/day to patients with MDS. In a particular embodiment, Compound 1provided herein can be administered in an amount of about 2 mg/day topatients with MDS. In a particular embodiment, Compound 1 providedherein can be administered in an amount of about 1 mg/day to patientswith MDS. In a particular embodiment, Compound 1 provided herein can beadministered in an amount of about 0.5 mg/day to patients with MDS.

In certain embodiments, the therapeutically or prophylacticallyeffective amount is from about 0.001 to about 100 mg/kg/day, from about0.01 to about 50 mg/kg/day, from about 0.01 to about 25 mg/kg/day, fromabout 0.01 to about 10 mg/kg/day, from about 0.01 to about 9 mg/kg/day,0.01 to about 8 mg/kg/day, from about 0.01 to about 7 mg/kg/day, fromabout 0.01 to about 6 mg/kg/day, from about 0.01 to about 5 mg/kg/day,from about 0.01 to about 4 mg/kg/day, from about 0.01 to about 3mg/kg/day, from about 0.01 to about 2 mg/kg/day, from about 0.01 toabout 1 mg/kg/day, or from about 0.01 to about 0.05 mg/kg/day.

The administered dose can also be expressed in units other thanmg/kg/day. For example, doses for parenteral administration can beexpressed as mg/m²/day. One of ordinary skill in the art would readilyknow how to convert doses from mg/kg/day to mg/m²/day to given eitherthe height or weight of a subject or both (see,www.fda.gov/cder/cancer/animalframe.htm). For example, a dose of 1mg/kg/day for a 65 kg human is approximately equal to 38 mg/m²/day.

In certain embodiments, the amount of Compound 1 administered issufficient to provide a plasma concentration of the compound at steadystate, ranging from about 0.001 to about 500 μM, about 0.002 to about200 μM, about 0.005 to about 100 μM, about 0.01 to about 50 μM, fromabout 1 to about 50 μM, about 0.02 to about 25 μM, from about 0.05 toabout 20 μM, from about 0.1 to about 20 μM, from about 0.5 to about 20μM, or from about 1 to about 20 μM.

In other embodiments, the amount of a lyophilized formulation ofCompound 1 administered is sufficient to provide a plasma concentrationof the compound at steady state, ranging from about 5 to about 100 nM,about 5 to about 50 nM, about 10 to about 100 nM, about 10 to about 50nM or from about 50 to about 100 nM.

As used herein, the term “plasma concentration at steady state” is theconcentration reached after a period of administration of a lyophilizedformulation provided herein. Once steady state is reached, there areminor peaks and troughs on the time dependent curve of the plasmaconcentration of the solid form.

In certain embodiments, the amount of a lyophilized formulation ofCompound 1 administered is sufficient to provide a maximum plasmaconcentration (peak concentration) of the compound, ranging from about0.001 to about 500 μM, about 0.002 to about 200 μM, about 0.005 to about100 μM, about 0.01 to about 50 μM, from about 1 to about 50 μM, about0.02 to about 25 μM, from about 0.05 to about 20 μM, from about 0.1 toabout 20 μM, from about 0.5 to about 20 μM, or from about 1 to about 20μM.

In certain embodiments, the amount of a lyophilized formulation ofCompound 1 administered is sufficient to provide a minimum plasmaconcentration (trough concentration) of the compound, ranging from about0.001 to about 500 μM, about 0.002 to about 200 μM, about 0.005 to about100 μM, about 0.01 to about 50 μM, from about 1 to about 50 μM, about0.01 to about 25 μM, from about 0.01 to about 20 μM, from about 0.02 toabout 20 μM, from about 0.02 to about 20 μM, or from about 0.01 to about20 μM.

In certain embodiments, the amount of a lyophilized formulation ofCompound 1 administered is sufficient to provide an area under the curve(AUC) of the compound, ranging from about 100 to about 100,000 ng*hr/mL,from about 1,000 to about 50,000 ng*hr/mL, from about 5,000 to about25,000 ng*hr/mL, or from about 5,000 to about 10,000 ng*hr/mL.

In certain embodiments, the patient to be treated with one of themethods provided herein has not been treated with anticancer therapyprior to the administration of a lyophilized formulation of Compound 1provided herein. In certain embodiments, the patient to be treated withone of the methods provided herein has been treated with anticancertherapy prior to the administration of a lyophilized formulation ofCompound 1 provided herein. In certain embodiments, the patient to betreated with one of the methods provided herein has developed drugresistance to the anticancer therapy.

The methods provided herein encompass treating a patient regardless ofpatient's age, although some diseases or disorders are more common incertain age groups.

The lyophilized formulation of Compound 1 provided herein can bedelivered as a single dose such as, e.g., a single bolus injection, orover time, such as, e.g., continuous infusion over time or divided bolusdoses over time. The lyophilized formulation of Compound 1 can beadministered repeatedly if necessary, for example, until the patientexperiences stable disease or regression, or until the patientexperiences disease progression or unacceptable toxicity. For example,stable disease for solid tumors generally means that the perpendiculardiameter of measurable lesions has not increased by 25% or more from thelast measurement. Response Evaluation Criteria in Solid Tumors (RECIST)Guidelines, Journal of the National Cancer Institute 92(3): 205-216(2000). Stable disease or lack thereof is determined by methods known inthe art such as evaluation of patient symptoms, physical examination,visualization of the tumor that has been imaged using X-ray, CAT, PET,or MRI scan and other commonly accepted evaluation modalities.

The a lyophilized formulation of Compound 1 provided herein can beadministered once daily (QD), or divided into multiple daily doses suchas twice daily (BID), three times daily (TID), and four times daily(QID). In addition, the administration can be continuous (i.e., dailyfor consecutive days or every day), intermittent, e.g., in cycles (i.e.,including days, weeks, or months of rest without drug). As used herein,the term “daily” is intended to mean that a therapeutic compound, isadministered once or more than once each day, for example, for a periodof time. The term “continuous” is intended to mean that a therapeuticcompound, is administered daily for an uninterrupted period of at least10 days to 52 weeks. The term “intermittent” or “intermittently” as usedherein is intended to mean stopping and starting at either regular orirregular intervals. For example, intermittent administration of thelyophilized formulation of Compound 1 is administration for one to sixdays per week, administration in cycles (e.g., daily administration forone to ten consecutive days of a 28 day cycle, then a rest period withno administration for rest of the 28 day cycle or daily administrationfor two to eight consecutive weeks, then a rest period with noadministration for up to one week), or administration on alternate days.The term “cycling” as used herein is intended to mean that a therapeuticcompound is administered daily or continuously but with a rest period.In some such embodiments, administration is once a day for two to sixdays, then a rest period with no administration for five to seven days.In some other such embodiments, administration is once a day for thefirst two to five or ten days of a 28 day cycle, followed by a restperiod with no administration for the rest of the 28 day cycle.

In some embodiments, the frequency of administration is in the range ofabout a daily dose to about a monthly dose. In certain embodiments,administration is once a day, twice a day, three times a day, four timesa day, once every other day, twice a week, once every week, once everytwo weeks, once every three weeks, or once every four weeks. In oneembodiment, Compound 1 is administered once a day. In anotherembodiment, Compound 1 is administered twice a day. In yet anotherembodiment, Compound 1 provided herein is administered three times aday. In still another embodiment, Compound 1 provided herein isadministered four times a day.

In certain embodiments, a lyophilized formulation of Compound 1 providedherein is administered once per day from one day to six months, from oneweek to three months, from one week to four weeks, from one week tothree weeks, or from one week to two weeks. In certain embodiments, alyophilized formulation of Compound 1 provided herein is administeredonce per day for one week, two weeks, three weeks, or four weeks. In oneembodiment, a lyophilized formulation of Compound 1 provided herein isadministered once per day for 1 day. In one embodiment, a lyophilizedformulation of Compound 1 provided herein is administered once per dayfor 2 days. In one embodiment, a lyophilized formulation of Compound 1provided herein is administered once per day for 3 days. In oneembodiment, a lyophilized formulation of Compound 1 provided herein isadministered once per day for 4 days. In one embodiment, a lyophilizedformulation of Compound 1 provided herein is administered once per dayfor 5 days. In one embodiment, a lyophilized formulation of Compound 1provided herein is administered once per day for 6 days. In oneembodiment, a lyophilized formulation of Compound 1 provided herein isadministered once per day for one week. In one embodiment, a lyophilizedformulation of Compound 1 provided herein is administered once per dayfor up to 10 days. In another embodiment, a lyophilized formulation ofCompound 1 provided herein is administered once per day for two weeks.In yet another embodiment, a lyophilized formulation of Compound 1provided herein is administered once per day for three weeks. In stillanother embodiment, a lyophilized formulation of Compound 1 providedherein is administered once per day for four weeks.

6.6.1 Combination Therapy

The lyophilized formulation of Compound 1 provided herein can also becombined or used in combination with other therapeutic agents useful inthe treatment and/or prevention of cancer described herein.

In one embodiment, provided herein is a method of treating, preventing,and/or managing cancer, comprising administering to a patient alyophilized formulation of Compound 1 provided herein in combinationwith one or more second active agents, and optionally in combinationwith radiation therapy, blood transfusions, or surgery. Examples ofsecond active agents are disclosed herein.

A lyophilized formulation of Compound 1 provided herein can also becombined or used in combination with other therapeutic agents useful inthe treatment and/or prevention of MDS described herein.

In one embodiment, provided herein is a method of treating, preventing,and/or managing MDS, comprising administering to a patient a lyophilizedformulation of Compound 1 provided herein in combination with one ormore second active agents, and optionally in combination with radiationtherapy, blood transfusions, or surgery. Examples of second activeagents are disclosed herein.

As used herein, the term “in combination” includes the use of more thanone therapy (e.g., one or more prophylactic and/or therapeutic agents).However, the use of the term “in combination” does not restrict theorder in which therapies (e.g., prophylactic and/or therapeutic agents)are administered to a patient with a disease or disorder. A firsttherapy (e.g., a prophylactic or therapeutic agent such as a lyophilizedformulation of Compound 1 provided herein, can be administered prior to(e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeksbefore), concomitantly with, or subsequent to (e.g., 5 minutes, 15minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks,4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) theadministration of a second therapy (e.g., a prophylactic or therapeuticagent) to the subject. Triple therapy is also contemplated herein.

Administration of a lyophilized formulation of Compound 1 providedherein and one or more second active agents to a patient can occursimultaneously or sequentially by the same or different routes ofadministration. The suitability of a particular route of administrationemployed for a particular active agent will depend on the active agentitself (e.g., whether it can be administered orally without decomposingprior to entering the blood stream) and the cancer being treated.

The route of administration of a lyophilized formulation of Compound 1is independent of the route of administration of a second therapy. Thus,in accordance with these embodiments, a lyophilized formulation ofCompound 1 is administered intravenously, and the second therapy can beadministered orally, parenterally, intraperitoneally, intravenously,intraarterially, transdermally, sublingually, intramuscularly, rectally,transbuccally, intranasally, liposomally, via inhalation, vaginally,intraoccularly, via local delivery by catheter or stent, subcutaneously,intraadiposally, intraarticularly, intrathecally, or in a slow releasedosage form.

In one embodiment, a lyophilized formulation of Compound 1 and a secondtherapy are administered by the same mode of administration, by IV. Inanother embodiment, a lyophilized formulation of Compound 1 isadministered by one mode of administration, e.g., by IV, whereas thesecond agent (an anticancer agent) is administered by another mode ofadministration, e.g., orally.

In one embodiment, the second active agent is administered intravenouslyor subcutaneously and once or twice daily in an amount of from about 1to about 1000 mg, from about 5 to about 500 mg, from about 10 to about350 mg, or from about 50 to about 200 mg. The specific amount of thesecond active agent will depend on the specific agent used, the type ofdisease being treated and/or managed, the severity and stage of disease,and the amount of Compound 1 and any optional additional active agentsconcurrently administered to the patient.

One or more second active ingredients or agents can be used togetherwith a lyophilized formulation of Compound 1 in the methods andcompositions provided herein. Second active agents can be largemolecules (e.g., proteins) or small molecules (e.g., syntheticinorganic, organometallic, or organic molecules).

Examples of large molecule active agents include, but are not limitedto, hematopoietic growth factors, cytokines, and monoclonal andpolyclonal antibodies, particularly, therapeutic antibodies to cancerantigens. Typical large molecule active agents are biological molecules,such as naturally occurring or synthetic or recombinant proteins.Proteins that are particularly useful in the methods and compositionsprovided herein include proteins that stimulate the survival and/orproliferation of hematopoietic precursor cells and immunologicallyactive poietic cells in vitro or in vivo. Other useful proteinsstimulate the division and differentiation of committed erythroidprogenitors in cells in vitro or in vivo. Particular proteins include,but are not limited to: interleukins, such as IL-2 (includingrecombinant IL-II (“rIL2”) and canarypox IL-2), IL-10, IL-12, and IL-18;interferons, such as interferon alfa-2a, interferon alfa-2b, interferonalfa-n1, interferon alfa-n3, interferon beta-I a, and interferon gamma-Ib; GM-CF and GM-CSF; and EPO.

In certain embodiments, GM-CSF, G-CSF, SCF or EPO is administeredsubcutaneously during about five days in a four or six week cycle in anamount ranging from about 1 to about 750 mg/m²/day, from about 25 toabout 500 mg/m²/day, from about 50 to about 250 mg/m²/day, or from about50 to about 200 mg/m²/day. In certain embodiments, GM-CSF may beadministered in an amount of from about 60 to about 500 mcg/m²intravenously over 2 hours or from about 5 to about 12 mcg/m²/daysubcutaneously. In certain embodiments, G-CSF may be administeredsubcutaneously in an amount of about 1 mcg/kg/day initially and can beadjusted depending on rise of total granulocyte counts. The maintenancedose of G-CSF may be administered in an amount of about 300 (in smallerpatients) or 480 mcg subcutaneously. In certain embodiments, EPO may beadministered subcutaneously in an amount of 10,000 Unit 3 times perweek.

Particular proteins that can be used in the methods and compositionsinclude, but are not limited to: filgrastim, which is sold in the UnitedStates under the trade name Neupogen® (Amgen, Thousand Oaks, Calif.);sargramostim, which is sold in the United States under the trade nameLeukine® (Immunex, Seattle, Wash.); and recombinant EPO, which is soldin the United States under the trade name Epogen® (Amgen, Thousand Oaks,Calif.).

Recombinant and mutated forms of GM-CSF can be prepared as described inU.S. Pat. Nos. 5,391,485; 5,393,870; and 5,229,496; all of which areincorporated herein by reference. Recombinant and mutated forms of G-CSFcan be prepared as described in U.S. Pat. Nos. 4,810,643; 4,999,291;5,528,823; and 5,580,755; the entireties of which are incorporatedherein by reference.

Also provided for use in combination with a lyophilized formulation ofCompound 1 provided herein are native, naturally occurring, andrecombinant proteins. Further encompassed are mutants and derivatives(e.g., modified forms) of naturally occurring proteins that exhibit, invivo, at least some of the pharmacological activity of the proteins uponwhich they are based. Examples of mutants include, but are not limitedto, proteins that have one or more amino acid residues that differ fromthe corresponding residues in the naturally occurring forms of theproteins. Also encompassed by the term “mutants” are proteins that lackcarbohydrate moieties normally present in their naturally occurringforms (e.g., nonglycosylated forms). Examples of derivatives include,but are not limited to, pegylated derivatives and fusion proteins, suchas proteins formed by fusing IgG1 or IgG3 to the protein or activeportion of the protein of interest. See, e.g., Penichet, M. L. andMorrison, S. L., J. Immunol. Methods 248:91-101 (2001).

Antibodies that can be used in combination with a lyophilizedformulation of Compound 1 provided herein include monoclonal andpolyclonal antibodies. Examples of antibodies include, but are notlimited to, trastuzumab (Herceptin®), rituximab (Rituxan®), bevacizumab(Avastin™), pertuzumab (Omnitarg™), tositumomab (Bexxar®), edrecolomab(Panorex®), and G250. The lyophilized formulation of Compound 1 can alsobe combined with, or used in combination with, anti-TNF-α antibodies,and/or anti-EGFR antibodies, such as, for example, Erbitux® orpanitumumab.

Large molecule active agents may be administered in the form ofanti-cancer vaccines. For example, vaccines that secrete, or cause thesecretion of, cytokines such as IL-2, G-CSF, and GM-CSF can be used inthe methods and pharmaceutical compositions provided. See, e.g., Emens,L. A., et al., Curr. Opinion Mol. Ther. 3(1):77-84 (2001).

Second active agents that are small molecules can also be used toalleviate adverse effects associated with the administration of alyophilized formulation of Compound 1 provided herein. However, likesome large molecules, many are believed to be capable of providing asynergistic effect when administered with (e.g., before, after orsimultaneously) a lyophilized formulation of Compound 1 provided herein.Examples of small molecule second active agents include, but are notlimited to, anti-cancer agents, antibiotics, immunosuppressive agents,and steroids.

In certain embodiments, the second agent is an HSP inhibitor, aproteasome inhibitor, a FLT3 inhibitior or a TOR kinase inhibitor.

Examples of anti-cancer agents to be used within the methods orcompositions described herein include, but are not limited to: acivicin;aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin;altretamine; ambomycin; ametantrone acetate; amsacrine; anastrozole;anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin;batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafidedimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine;busulfan; cactinomycin; calusterone; caracemide; carbetimer;carboplatin; carmustine; carubicin hydrochloride; carzelesin;cedefingol; celecoxib (COX-2 inhibitor); chlorambucil; cirolemycin;cisplatin; cladribine; clofarabine; crisnatol mesylate;cyclophosphamide; Ara-C; dacarbazine; dactinomycin; daunorubicinhydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguaninemesylate; diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride;droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin;edatrexate; eflornithine hydrochloride; elsamitrucin; enloplatin;enpromate; epipropidine; epirubicin hydrochloride; erbulozole;esorubicin hydrochloride; estramustine; estramustine phosphate sodium;etanidazole; etoposide; etoposide phosphate; etoprine; fadrozolehydrochloride; fazarabine; fenretinide; floxuridine; fludarabinephosphate; fluorouracil; flurocitabine; fosquidone; fostriecin sodium;gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicinhydrochloride; ifosfamide; ilmofosine; iproplatin; irinotecan;irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolideacetate; liarozole hydrochloride; lometrexol sodium; lomustine;losoxantrone hydrochloride; masoprocol; maytansine; mechlorethaminehydrochloride; megestrol acetate; melengestrol acetate; melphalan;menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine;meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin;mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolicacid; nocodazole; nogalamycin; omacetaxine; ormaplatin; oxisuran;paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate;perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride;plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine;procarbazine hydrochloride; puromycin; puromycin hydrochloride;pyrazofurin; riboprine; safingol; safingol hydrochloride; semustine;simtrazene; sorafenib; sparfosate sodium; sparsomycin; spirogermaniumhydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin;sulofenur; talisomycin; tecogalan sodium; taxotere; tegafur;teloxantrone hydrochloride; temoporfin; teniposide; teroxirone;testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin;tirapazamine; toremifene citrate; trestolone acetate; triciribinephosphate; trimetrexate; trimetrexate glucuronate; triptorelin;tubulozole hydrochloride; uracil mustard; uredepa; vapreotide;verteporfin; vinblastine sulfate; vincristine sulfate; vindesine;vindesine sulfate; vinepidine sulfate; vinglycinate sulfate;vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate;vinzolidine sulfate; vorozole; zeniplatin; zinostatin; and zorubicinhydrochloride.

Other anti-cancer drugs to be included within the methods orcompositions include, but are not limited to: 20-epi-1,25dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin;acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists;altretamine; ambamustine; amidox; amifostine; aminolevulinic acid;amrubicin; amsacrine; anagrelide; anastrozole; andrographolide;angiogenesis inhibitors; antagonist D; antagonist G; antarelix;anti-dorsalizing morphogenetic protein-1; antiandrogen, prostaticcarcinoma; antiestrogen; antineoplaston; antisense oligonucleotides;aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators;apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine;atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3;azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol;batimastat; BCR/ABL antagonists; benzochlorins; benzoylstaurosporine;beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid;bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine;bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane;buthionine sulfoximine; calcipotriol; calphostin C; camptothecinderivatives; capecitabine; carboxamide-amino-triazole;carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor;carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropinB; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost;cis-porphyrin; cladribine; clomifene analogues; clotrimazole;collismycin A; collismycin B; combretastatin A4; combretastatinanalogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8;cryptophycin A derivatives; curacin A; cyclopentanthraquinones;cycloplatam; cypemycin; Ara-C ocfosfate; cytolytic factor; cytostatin;dacliximab; decitabine; dehydrodidemnin B; deslorelin; dexamethasone;dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox;diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin;diphenyl spiromustine; docetaxel; docosanol; dolasetron; doxifluridine;doxorubicin; droloxifene; dronabinol; duocarmycin SA; ebselen;ecomustine; edelfosine; edrecolomab; eflornithine; elemene; emitefur;epirubicin; epristeride; estramustine analogue; estrogen agonists;estrogen antagonists; etanidazole; etoposide phosphate; exemestane;fadrozole; fazarabine; fenretinide; filgrastim; finasteride;flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicinhydrochloride; forfenimex; formestane; fostriecin; fotemustine;gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix;gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam;heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid;idarubicin; idoxifene; idramantone; ilmofosine; ilomastat; imatinib(e.g., Gleevec®); imiquimod; immunostimulant peptides; insulin-likegrowth factor-1 receptor inhibitor; interferon agonists; interferons;interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact;irsogladine; isobengazole; isohomohalicondrin B; itasetron;jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;leukemia inhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; loxoribine; lurtotecan; lutetiumtexaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A;marimastat; masoprocol; maspin; matrilysin inhibitors; matrixmetalloproteinase inhibitors; menogaril; merbarone; meterelin;methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine;mirimostim; mitoguazone; mitolactol; mitomycin analogues; mitonafide;mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene;molgramostim; Erbitux, human chorionic gonadotrophin; monophosphoryllipid A+myobacterium cell wall sk; mopidamol; mustard anticancer agent;mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; nilutamide; nisamycin; nitric oxidemodulators; nitroxide antioxidant; nitrullyn; oblimersen (Genasense®);O⁶-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone;ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin;osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues;paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid;panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rohitukine;romurtide; roquinimex; rubiginone B1; ruboxyl; safingol; saintopin;SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine;senescence derived inhibitor 1; sense oligonucleotides; signaltransduction inhibitors; sizofiran; sobuzoxane; sodium borocaptate;sodium phenylacetate; solverol; somatomedin binding protein; sonermin;sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin1; squalamine; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; tallimustine; tamoxifen methiodide; tauromustine;tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomeraseinhibitors; temoporfin; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenebichloride; topsentin; toremifene; translation inhibitors; tretinoin;triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron;turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors;ubenimex; urogenital sinus-derived growth inhibitory factor; urokinasereceptor antagonists; vapreotide; variolin B; velaresol; veramine;verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole;zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer.

In certain embodiments, a lyophilized formulation of Compound 1 isadministered in combination with checkpoint inhibitors. In oneembodiment, one checkpoint inhibitor is used in combination with alyophilized formulation of Compound 1 in connection with the methodsprovided herein. In another embodiment, two checkpoint inhibitors areused in combination with a lyophilized formulation of Compound 1 inconnection with the methods provided herein. In yet another embodiment,three or more checkpoint inhibitors are used in combination with alyophilized formulation of Compound 1 in connection with the methodsprovided herein.

As used herein, the term “immune checkpoint inhibitor” or “checkpointinhibitor” refers to molecules that totally or partially reduce,inhibit, interfere with or modulate one or more checkpoint proteins.Without being limited by a particular theory, checkpoint proteinsregulate T-cell activation or function. Numerous checkpoint proteins areknown, such as CTLA-4 and its ligands CD80 and CD86; and PD-1 with itsligands PD-L1 and PD-L2 (Pardoll, Nature Reviews Cancer, 2012, 12,252-264). These proteins appear responsible for co-stimulatory orinhibitory interactions of T-cell responses. Immune checkpoint proteinsappear to regulate and maintain self-tolerance and the duration andamplitude of physiological immune responses. Immune checkpointinhibitors include antibodies or are derived from antibodies.

In one embodiment, the checkpoint inhibitor is a CTLA-4 inhibitor. Inone embodiment, the CTLA-4 inhibitor is an anti-CTLA-4 antibody.Examples of anti-CTLA-4 antibodies include, but are not limited to,those described in U.S. Pat. Nos. 5,811,097; 5,811,097; 5,855,887;6,051,227; 6,207,157; 6,682,736; 6,984,720; and 7,605,238, all of whichare incorporated herein in their entireties. In one embodiment, theanti-CTLA-4 antibody is tremelimumab (also known as ticilimumab orCP-675,206). In another embodiment, the anti-CTLA-4 antibody isipilimumab (also known as MDX-010 or MDX-101). Ipilimumab is a fullyhuman monoclonal IgG antibody that binds to CTLA-4. Ipilimumab ismarketed under the trade name Yervoy™.

In one embodiment, the checkpoint inhibitor is a PD-1/PD-L1 inhibitor.Examples of PD-1/PD-L1 inhibitors include, but are not limited to, thosedescribed in U.S. Pat. Nos. 7,488,802; 7,943,743; 8,008,449; 8,168,757;8,217,149, and PCT Patent Application Publication Nos. WO2003042402,WO2008156712, WO2010089411, WO2010036959, WO2011066342, WO2011159877,WO2011082400, and WO2011161699, all of which are incorporated herein intheir entireties.

In one embodiment, the checkpoint inhibitor is a PD-1 inhibitor. In oneembodiment, the PD-1 inhibitor is an anti-PD-1 antibody. In oneembodiment, the anti-PD-1 antibody is nivolumab (also known as ONO-4538,BMS-936558, or MDX1106) or pembrolizumab (also known as MK-3475, SCH900475, or lambrolizumab). In one embodiment, the anti-PD-1 antibody isnivolumab. Nivolumab is a human IgG4 anti-PD-1 monoclonal antibody, andis marketed under the trade name Opdivo™. In another embodiment, theanti-PD-1 antibody is pembrolizumab. Pembrolizumab is a humanizedmonoclonal IgG4 antibody and is marketed under the trade name Keytruda™.In yet another embodiment, the anti-PD-1 antibody is CT-011, a humanizedantibody. CT-011 administered alone has failed to show response intreating acute myeloid leukemia (AML) at relapse. In yet anotherembodiment, the anti-PD-1 antibody is AMP-224, a fusion protein.

In one embodiment, the checkpoint inhibitor is a PD-L1 inhibitor. In oneembodiment, the PD-L1 inhibitor is an anti-PD-L1 antibody. In oneembodiment, the anti-PD-L1 antibody is MEDI4736 (durvalumab). In anotherembodiment, the anti-PD-L1 antibody is BMS-936559 (also known asMDX-1105-01). In yet another embodiment, the PD-L1 inhibitor isatezolizumab (also known as MPDL3280A, and Tecentriq®).

In one embodiment, the checkpoint inhibitor is a PD-L2 inhibitor. In oneembodiment, the PD-L2 inhibitor is an anti-PD-L2 antibody. In oneembodiment, the anti-PD-L2 antibody is rHIgM12B7A.

In one embodiment, the checkpoint inhibitor is a lymphocyte activationgene-3 (LAG-3) inhibitor. In one embodiment, the LAG-3 inhibitor isIMP321, a soluble Ig fusion protein (Brignone et al., J. Immunol., 2007,179, 4202-4211). In another embodiment, the LAG-3 inhibitor isBMS-986016.

In one embodiment, the checkpoint inhibitors is a B7 inhibitor. In oneembodiment, the B7 inhibitor is a B7-H3 inhibitor or a B7-H4 inhibitor.In one embodiment, the B7-H3 inhibitor is MGA271, an anti-B7-H3 antibody(Loo et al., Clin. Cancer Res., 2012, 3834).

In one embodiment, the checkpoint inhibitors is a TIM3 (T-cellimmunoglobulin domain and mucin domain 3) inhibitor (Fourcade et al., J.Exp. Med., 2010, 207, 2175-86; Sakuishi et al., J. Exp. Med., 2010, 207,2187-94).

In one embodiment, the checkpoint inhibitor is an OX40 (CD134) agonist.In one embodiment, the checkpoint inhibitor is an anti-OX40 antibody. Inone embodiment, the anti-OX40 antibody is anti-OX-40. In anotherembodiment, the anti-OX40 antibody is MEDI6469.

In one embodiment, the checkpoint inhibitor is a GITR agonist. In oneembodiment, the checkpoint inhibitor is an anti-GITR antibody. In oneembodiment, the anti-GITR antibody is TRX518.

In one embodiment, the checkpoint inhibitor is a CD137 agonist. In oneembodiment, the checkpoint inhibitor is an anti-CD137 antibody. In oneembodiment, the anti-CD137 antibody is urelumab. In another embodiment,the anti-CD137 antibody is PF-05082566.

In one embodiment, the checkpoint inhibitor is a CD40 agonist. In oneembodiment, the checkpoint inhibitor is an anti-CD40 antibody. In oneembodiment, the anti-CD40 antibody is CF-870,893.

In one embodiment, the checkpoint inhibitor is recombinant humaninterleukin-15 (rhIL-15).

In one embodiment, the checkpoint inhibitor is an IDO inhibitor. In oneembodiment, the IDO inhibitor is INCB024360. In another embodiment, theIDO inhibitor is indoximod.

In certain embodiments, the combination therapies provided hereininclude two or more of the checkpoint inhibitors described herein(including checkpoint inhibitors of the same or different class).Moreover, the combination therapies described herein can be used incombination with second active agents as described herein whereappropriate for treating diseases described herein and understood in theart.

In certain embodiments, Compound 1 can be used in combination with oneor more immune cells expressing one or more chimeric antigen receptors(CARs) on their surface (e.g., a modified immune cell). Generally, CARscomprise an extracellular domain from a first protein e.g., anantigen-binding protein), a transmembrane domain, and an intracellularsignaling domain. In certain embodiments, once the extracellular domainbinds to a target protein such as a tumor-associated antigen (TAA) ortumor-specific antigen (TSA), a signal is generated via theintracellular signaling domain that activates the immune cell, e.g., totarget and kill a cell expressing the target protein.

Extracellular domains: The extracellular domains of the CARs bind to anantigen of interest. In certain embodiments, the extracellular domain ofthe CAR comprises a receptor, or a portion of a receptor, that binds tosaid antigen. In certain embodiments, the extracellular domaincomprises, or is, an antibody or an antigen-binding portion thereof. Inspecific embodiments, the extracellular domain comprises, or is, asingle chain Fv (scFv) domain. The single-chain Fv domain can comprise,for example, a V_(L) linked to V_(H) by a flexible linker, wherein saidV_(L) and V_(H) are from an antibody that binds said antigen.

In certain embodiments, the antigen recognized by the extracellulardomain of a polypeptide described herein is a tumor-associated antigen(TAA) or a tumor-specific antigen (TSA). In various specificembodiments, the tumor-associated antigen or tumor-specific antigen is,without limitation, Her2, prostate stem cell antigen (PSCA),alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancerantigen-125 (CA-125), CA19-9, calretinin, MUC-1, B cell maturationantigen (BCMA), epithelial membrane protein (EMA), epithelial tumorantigen (ETA), tyrosinase, melanoma-24 associated antigen (MAGE), CD19,CD22, CD27, CD30, CD34, CD45, CD70, CD99, CD117, EGFRvIII (epidermalgrowth factor variant III), mesothelin, PAP (prostatic acidphosphatase), prostein, TARP (T cell receptor gamma alternate readingframe protein), Trp-p8, STEAPI (six-transmembrane epithelial antigen ofthe prostate 1), chromogranin, cytokeratin, desmin, glial fibrillaryacidic protein (GFAP), gross cystic disease fluid protein (GCDFP-15),HMB-45 antigen, protein melan-A (melanoma antigen recognized by Tlymphocytes; MART-I), myo-D1, muscle-specific actin (MSA),neurofilament, neuron-specific enolase (NSE), placental alkalinephosphatase, synaptophysis, thyroglobulin, thyroid transcriptionfactor-1, the dimeric form of the pyruvate kinase isoenzyme type M2(tumor M2-PK), an abnormal ras protein, or an abnormal p53 protein. Incertain other embodiments, the TAA or TSA recognized by theextracellular domain of a CAR is integrin αvβ33 (CD61), galactin, orRal-B.

In certain embodiments, the TAA or TSA recognized by the extracellulardomain of a CAR is a cancer/testis (CT) antigen, e.g., BAGE, CAGE,CTAGE, FATE, GAGE, HCA661, HOM-TES-85, MAGEA, MAGEB, MAGEC, NA88,NY-ES0-1, NY-SAR-35, OY-TES-1, SPANXBI, SPA17, SSX, SYCPI, or TPTE.

In certain other embodiments, the TAA or TSA recognized by theextracellular domain of a CAR is a carbohydrate or ganglioside, e.g.,fuc-GMI, GM2 (oncofetal antigen-immunogenic-1; OFA-I-1); GD2 (OFA-I-2),GM3, GD3, and the like.

In certain other embodiments, the TAA or TSA recognized by theextracellular domain of a CAR is alpha-actinin-4, Bage-1, BCR-ABL,Bcr-Abl fusion protein, beta-catenin, CA 125, CA 15-3 (CA 27.29\BCAA),CA 195, CA 242, CA-50, CAM43, Casp-8, cdc27, cdk4, cdkn2a, CEA, coa-1,dek-can fusion protein, EBNA, EF2, Epstein Barr virus antigens,ETV6-AML1 fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAA0205, Mart2,Mum-1, 2, and 3, neo-PAP, myosin class I, OS-9, pml-RARα fusion protein,PTPRK, K-ras, N-ras, triosephosphate isomerase, Gage 3,4,5,6,7, GnTV,Herv-K-mel, Lage-1, NA-88, NY-Eso-1/Lage-2, SP17, SSX-2, TRP2-Int2,gp100 (Pmel17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, RAGE, GAGE-1,GAGE-2, p15(58), RAGE, SCP-1, Hom/Mel-40, PRAME, p53, HRas, HER-2/neu,E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, human papillomavirus (HPV) antigensE6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3,c-met, nm-23H1, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras,13-Catenin, Mum-1, p16, TAGE, PSMA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, 13HCG, BCA225, BTAA, CD68\KP1, C0-029, FGF-5, G250, Ga733(EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\70K, NY-C0-1, RCAS1,SDCCAG16, TA-90, TAAL6, TAG72, TLP, or TPS.

In various specific embodiments, the tumor-associated antigen ortumor-specific antigen is an AML-related tumor antigens, as described inS. Anguille et al, Leukemia (2012), 26, 2186-2196.

Other tumor-associated and tumor-specific antigens are known to those inthe art.

Receptors, antibodies, and scFvs that bind to TSAs and TAAs, useful inconstructing chimeric antigen receptors, are known in the art, as arenucleotide sequences that encode them.

In certain specific embodiments, the antigen recognized by theextracellular domain of a chimeric antigen receptor is an antigen notgenerally considered to be a TSA or a TAA, but which is neverthelessassociated with tumor cells, or damage caused by a tumor. In certainembodiments, for example, the antigen is, e.g., a growth factor,cytokine or interleukin, e.g., a growth factor, cytokine, or interleukinassociated with angiogenesis or vasculogenesis. Such growth factors,cytokines, or interleukins can include, e.g., vascular endothelialgrowth factor (VEGF), basic fibroblast growth factor (bFGF),platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF),insulin-like growth factor (IGF), or interleukin-8 (IL-8). Tumors canalso create a hypoxic environment local to the tumor. As such, in otherspecific embodiments, the antigen is a hypoxia-associated factor, e.g.,HIF-1α, HIF-1β, HIF-2α, HIF-2β, HIF-3α, or HIF-3β. Tumors can also causelocalized damage to normal tissue, causing the release of moleculesknown as damage associated molecular pattern molecules (DAMPs; alsoknown as alarmins). In certain other specific embodiments, therefore,the antigen is a DAMP, e.g., a heat shock protein, chromatin-associatedprotein high mobility group box 1 (HMGB 1), S100A8 (MRP8, calgranulinA), S100A9 (MRP14, calgranulin B), serum amyloid A (SAA), or can be adeoxyribonucleic acid, adenosine triphosphate, uric acid, or heparinsulfate.

Transmembrane domain: In certain embodiments, the extracellular domainof the CAR is joined to the transmembrane domain of the polypeptide by alinker, spacer or hinge polypeptide sequence, e.g., a sequence from CD28or a sequence from CTLA4. The transmembrane domain can be obtained orderived from the transmembrane domain of any transmembrane protein, andcan include all or a portion of such transmembrane domain. In specificembodiments, the transmembrane domain can be obtained or derived from,e.g., CD8, CD16, a cytokine receptor, and interleukin receptor, or agrowth factor receptor, or the like.

Intracellular signaling domains: In certain embodiments, theintracellular domain of a CAR is or comprises an intracellular domain ormotif of a protein that is expressed on the surface of T cells andtriggers activation and/or proliferation of said T cells. Such a domainor motif is able to transmit a primary antigen-binding signal that isnecessary for the activation of a T lymphocyte in response to theantigen's binding to the CAR's extracellular portion. Typically, thisdomain or motif comprises, or is, an ITAM (immunoreceptor tyrosine-basedactivation motif). ITAM-containing polypeptides suitable for CARsinclude, for example, the zeta CD3 chain (CD3ζ) or ITAM-containingportions thereof. In a specific embodiment, the intracellular domain isa CD3ζ intracellular signaling domain. In other specific embodiments,the intracellular domain is from a lymphocyte receptor chain, a TCR/CD3complex protein, an Fe receptor subunit or an IL-2 receptor subunit. Incertain embodiments, the CAR additionally comprises one or moreco-stimulatory domains or motifs, e.g., as part of the intracellulardomain of the polypeptide. The one or more co-stimulatory domains ormotifs can be, or can comprise comprise, one or more of a co-stimulatoryCD27 polypeptide sequence, a co-stimulatory CD28 polypeptide sequence, aco-stimulatory OX40 (CD134) polypeptide sequence, a co-stimulatory 4-1BB(CD137) polypeptide sequence, or a co-stimulatory inducible T-cellcostimulatory (ICOS) polypeptide sequence, or other costimulatory domainor motif, or any combination thereof.

The CAR may also comprise a T cell survival motif. The T cell survivalmotif can be any polypeptide sequence or motif that facilitates thesurvival of the T lymphocyte after stimulation by an antigen. In certainembodiments, the T cell survival motif is, or is derived from, CD3,CD28, an intracellular signaling domain of IL-7 receptor (IL-7R), anintracellular signaling domain of IL-12 receptor, an intracellularsignaling domain of IL-15 receptor, an intracellular signaling domain ofIL-21 receptor, or an intracellular signaling domain of transforminggrowth factor β (TGFβ) receptor.

The modified immune cells expressing the CARs can be, e.g., Tlymphocytes (T cells, e.g., CD4+ T cells or CD8+ T cells), cytotoxiclymphocytes (CTLs) or natural killer (NK) cells. T lymphocytes used inthe compositions and methods provided herein may be naive T lymphocytesor MHC-restricted T lymphocytes. In certain embodiments, the Tlymphocytes are tumor infiltrating lymphocytes (TILs). In certainembodiments, the T lymphocytes have been isolated from a tumor biopsy,or have been expanded from T lymphocytes isolated from a tumor biopsy.In certain other embodiments, the T cells have been isolated from, orare expanded from T lymphocytes isolated from, peripheral blood, cordblood, or lymph. Immune cells to be used to generate modified immunecells expressing a CAR can be isolated using art-accepted, routinemethods, e.g., blood collection followed by apheresis and optionallyantibody-mediated cell isolation or sorting.

The modified immune cells are preferably autologous to an individual towhom the modified immune cells are to be administered. In certain otherembodiments, the modified immune cells are allogeneic to an individualto whom the modified immune cells are to be administered. Whereallogeneic T lymphocytes or NK cells are used to prepare modified Tlymphocytes, it is preferable to select T lymphocytes or NK cells thatwill reduce the possibility of graft-versus-host disease (GVHD) in theindividual. For example, in certain embodiments, virus-specific Tlymphocytes are selected for preparation of modified T lymphocytes; suchlymphocytes will be expected to have a greatly reduced native capacityto bind to, and thus become activated by, any recipient antigens. Incertain embodiments, recipient-mediated rejection of allogeneic Tlymphocytes can be reduced by co-administration to the host of one ormore immunosuppressive agents, e.g., cyclosporine, tacrolimus,sirolimus, cyclophosphamide, or the like.

T lymphocytes, e.g., unmodified T lymphocytes, or T lymphocytesexpressing CD3 and CD28, or comprising a polypeptide comprising a CD3ζsignaling domain and a CD28 co-stimulatory domain, can be expanded usingantibodies to CD3 and CD28, e.g., antibodies attached to beads; see,e.g., U.S. Pat. Nos. 5,948,893; 6,534,055; 6,352,694; 6,692,964;6,887,466; and 6,905,681.

The modified immune cells, e.g., modified T lymphocytes, can optionallycomprise a “suicide gene” or “safety switch” that enables killing ofsubstantially all of the modified immune cells when desired. Forexample, the modified T lymphocytes, in certain embodiments, cancomprise an HSV thymidine kinase gene (HSV-TK), which causes death ofthe modified T lymphocytes upon contact with gancyclovir. In anotherembodiment, the modified T lymphocytes comprise an inducible caspase,e.g., an inducible caspase 9 (icaspase9), e.g., a fusion protein betweencaspase 9 and human FK506 binding protein allowing for dimerizationusing a specific small molecule pharmaceutical. See Straathof et al.,Blood 105(11):4247-4254 (2005).

Specific second active agents particularly useful in the methods orcompositions include, but are not limited to, rituximab, oblimersen(Genasense®), remicade, docetaxel, celecoxib, melphalan, dexamethasone(Decadron®), steroids, gemcitabine, cisplatinum, temozolomide,etoposide, cyclophosphamide, temodar, carboplatin, procarbazine,gliadel, tamoxifen, topotecan, methotrexate, Arisa®, taxol, taxotere,fluorouracil, leucovorin, irinotecan, xeloda, interferon alpha,pegylated interferon alpha (e.g., PEG INTRON-A), capecitabine,cisplatin, thiotepa, fludarabine, carboplatin, liposomal daunorubicin,Ara-C, doxetaxol, pacilitaxel, vinblastine, IL-2, GM-CSF, dacarbazine,vinorelbine, zoledronic acid, palmitronate, biaxin, busulphan,prednisone, bisphosphonate, arsenic trioxide, vincristine, doxorubicin(Doxil®), paclitaxel, ganciclovir, adriamycin, estramustine sodiumphosphate (Emcyt®), sulindac, and etoposide.

In certain embodiments of the methods provided herein, use of a secondactive agent in combination with a lyophilized formulation of Compound 1provided herein may be modified or delayed during or shortly followingadministration of a lyophilized formulation of Compound 1 providedherein as deemed appropriate by the practitioner of skill in the art. Incertain embodiments, subjects being administered a lyophilizedformulation of Compound 1 provided herein alone or in combination withother therapies may receive supportive care including antiemetics,myeloid growth factors, and transfusions of platelets, when appropriate.In some embodiments, subjects being administered a lyophilizedformulation of Compound 1 provided herein may be administered a growthfactor as a second active agent according to the judgment of thepractitioner of skill in the art. In some embodiments, provided isadministration of a lyophilized formulation of Compound 1 providedherein in combination with erythropoietin or darbepoetin (Aranesp).

In certain embodiments, a lyophilized formulation of Compound 1 providedherein is administered with gemcitabine, cisplatinum, 5-fluorouracil,mitomycin, methotrexate, vinblastine, doxorubicin, carboplatin,thiotepa, paclitaxel or docetaxel to patients with locally advanced ormetastatic transitional cell bladder cancer.

In certain embodiments, a lyophilized formulation of Compound 1 providedherein is administered in combination with a second active ingredient asfollows: temozolomide to pediatric patients with relapsed or progressivebrain tumors or recurrent neuroblastoma; celecoxib, etoposide andcyclophosphamide for relapsed or progressive CNS cancer; temodar topatients with recurrent or progressive meningioma, malignant meningioma,hemangiopericytoma, multiple brain metastases, relapsed brain tumors, ornewly diagnosed glioblastoma multiforms; irinotecan to patients withrecurrent glioblastoma; carboplatin to pediatric patients with brainstem glioma; procarbazine to pediatric patients with progressivemalignant gliomas; cyclophosphamide to patients with poor prognosismalignant brain tumors, newly diagnosed or recurrent glioblastomamultiforms; Gliadel® for high grade recurrent malignant gliomas;temozolomide and tamoxifen for anaplastic astrocytoma; or topotecan forgliomas, glioblastoma, anaplastic astrocytoma or anaplasticoligodendroglioma.

In certain embodiments, a lyophilized formulation of Compound 1 providedherein is administered with methotrexate, cyclophosphamide,5-fluorouracil, taxane, everolimus, abraxane, lapatinib, herceptin,pamidronate disodium, eribulin mesylate, everolimus, gemcitabine,palbociclib, ixabepilone, kadcyla, pertuzumab, theotepa, aromataseinhibitors, exemestane, selective estrogen modulators, estrogen receptorantagonists, anthracyclines, emtansine, and/or pexidartinib to patientswith metastatic breast cancer.

In certain embodiments, a lyophilized formulation of Compound 1 providedherein is administered with temozolomide, doxorubicin (Adriamycin),fluorouracil (Adrucil, 5-fluorouracil), or streptozocin (Zanosar) topatients with neuroendocrine tumors.

In certain embodiments, a lyophilized formulation of Compound 1 providedherein is administered with methotrexate, gemcitabine, cisplatin,cetuximab, 5-fluorouracil, bleomycin, docetaxel or carboplatin topatients with recurrent or metastatic head or neck cancer.

In certain embodiments, a lyophilized formulation of Compound 1 providedherein is administered with gemcitabine, abraxane, 5-fluorouracil,afinitor, irinotecan, mitomycin C, sunitinib or tarceva to patients withpancreatic cancer.

In certain embodiments, a lyophilized formulation of Compound 1 providedherein is administered to patients with colon cancer in combination withARISA®, avastatin, oxaliplatin, 5-fluorouracil, irinotecan,capecitabine, cetuximab, ramucirumab, panitumumab, bevacizumab,leucovorin calcium, lonsurf, regorafenib, ziv-aflibercept, taxol, and/ortaxotere.

In certain embodiments, a lyophilized formulation of Compound 1 providedherein is administered with capecitabine and/or vemurafenib to patientswith refractory colorectal cancer or patients who fail first linetherapy or have poor performance in colon or rectal adenocarcinoma.

In certain embodiments, a lyophilized formulation of Compound 1 providedherein is administered in combination with fluorouracil, leucovorin, andirinotecan to patients with Dukes C & D colorectal cancer or to patientswho have been previously treated for metastatic colorectal cancer.

In certain embodiments, a lyophilized formulation of Compound 1 providedherein is administered to patients with refractory colorectal cancer incombination with capecitabine, xeloda, and/or irinotecan.

In certain embodiments, a lyophilized formulation of Compound 1 providedherein is administered with capecitabine and irinotecan to patients withrefractory colorectal cancer or to patients with unresectable ormetastatic colorectal carcinoma.

In certain embodiments, a lyophilized formulation of Compound 1 providedherein is administered alone or in combination with interferon alpha orcapecitabine to patients with unresectable or metastatic hepatocellularcarcinoma; or with cisplatin and thiotepa, or with sorafenib tosylate topatients with primary or metastatic liver cancer.

In certain embodiments, a lyophilized formulation of Compound 1 providedherein is administered in combination with doxorubicin, paclitaxel,vinblastine or pegylated interferon alpha to patients with Kaposi'ssarcoma.

In certain embodiments, a lyophilized formulation of Compound 1 providedherein is administered in combination with arsenic trioxide,fludarabine, carboplatin, daunorubicin, cyclophosphamide, cytarabine,doxorubicin, idarubicin, mitoxantrone hydrochloride, thioguanine,vincritine, and/or topotecan to patients with acute myeloid leukemia,including refractory or relapsed or high-risk acute myeloid leukemia.

In certain embodiments, a lyophilized formulation of Compound 1 providedherein is administered in combination with liposomal daunorubicin,topotecan and/or cytarabine to patients with unfavorable karotype acutemyeloblastic leukemia.

In certain embodiments, a lyophilized formulation of Compound 1 providedherein is administered in combination with methotrexate, mechlorethaminehydrochloride, afatinib dimaleate, pemetrexed, bevacizumab, carboplatin,cisplatin, ceritinib, crizotinib, ramucirumab, pembrolizumab, docetaxel,vinorelbine tartrate, gemcitabine, abraxane, erlotinib, geftinib, and/oririnotecan to patients with non-small cell lung cancer.

In certain embodiments, a lyophilized formulation of Compound 1 providedherein is administered in combination with carboplatin and irinotecan topatients with non-small cell lung cancer.

In certain embodiments, a lyophilized formulation of Compound 1 providedherein is administered with doxetaxol to patients with non-small celllung cancer who have been previously treated with carbo/etoposide andradiotherapy.

In certain embodiments, a lyophilized formulation of Compound 1 providedherein is administered in combination with carboplatin and/or taxotere,or in combination with carboplatin, pacilitaxel and/or thoracicradiotherapy to patients with non-small cell lung cancer.

In certain embodiments, a lyophilized formulation of Compound 1 providedherein is administered in combination with taxotere to patients withstage IIIB or IV non-small cell lung cancer.

In certain embodiments, a lyophilized formulation of Compound 1 providedherein is administered in combination with oblimersen)(Genasense®,methotrexate, mechlorethamine hydrochloride, etoposide, topotecan ordoxorubicin to patients with small cell lung cancer.

In certain embodiments, a lyophilized formulation of Compound 1 providedherein is administered in combination with ABT-737 (Abbott Laboratories)and/or obatoclax (GX15-070) to patients with lymphoma and other bloodcancers.

In certain embodiments, a lyophilized formulation of Compound 1 providedherein is administered alone or in combination with a second activeingredient such as vinblastine or fludarabine adcetris, ambochlorin,becenum, bleomycin, brentuximab vedotin, carmustinem chlorambucil,cyclophosphamide, dacarbazine, doxorubicin, lomustine, matulane,mechlorethamine hydrochloride, prednisone, procarbazine hydrochloride orvincristine to patients with various types of lymphoma, including, butnot limited to, Hodgkin's lymphoma, non-Hodgkin's lymphoma, cutaneousT-Cell lymphoma, cutaneous B-Cell lymphoma, diffuse large B-Celllymphoma or relapsed or refractory low grade follicular lymphoma.

In certain embodiments, a lyophilized formulation of Compound 1 providedherein is administered in combination with taxotere, dabrafenib,imlygic, ipilimumab, pembrolizumab, nivolumab, trametinib, vemurafenib,talimogene laherparepvec, IL-2, IFN, GM-CSF, and/or dacarbazine topatients with various types or stages of melanoma.

In certain embodiments, a lyophilized formulation of Compound 1 providedherein is administered alone or in combination with vinorelbine topatients with malignant mesothelioma, or stage IIIB non-small cell lungcancer with pleural implants or malignant pleural effusion mesotheliomasyndrome.

In certain embodiments, a lyophilized formulation of Compound 1 providedherein is administered to patients with various types or stages ofmultiple myeloma in combination with dexamethasone, zoledronic acid,palmitronate, GM-CSF, biaxin, vinblastine, melphalan, busulphan,cyclophosphamide, IFN, prednisone, bisphosphonate, celecoxib, arsenictrioxide, PEG INTRON-A, vincristine, becenum, bortezomib, carfilzomib,doxorubicin, panobinostat, lenalidomide, pomalidomide, thalidomide,mozobil or a combination thereof.

In certain embodiments, a lyophilized formulation of Compound 1 providedherein is administered to patients with various types or stages ofmultiple myeloma in combination with chimeric antigen receptor (CAR)T-cells.

In certain embodiments, a lyophilized formulation of Compound 1 providedherein is administered to patients with relapsed or refractory multiplemyeloma in combination with doxorubicin (Doxil®), vincristine and/ordexamethasone (Decadron®).

In certain embodiments, a lyophilized formulation of Compound 1 providedherein is administered to patients with various types or stages ofovarian cancer such as peritoneal carcinoma, papillary serous carcinoma,refractory ovarian cancer or recurrent ovarian cancer, in combinationwith taxol, carboplatin, doxorubicin, gemcitabine, cisplatin, xeloda,paclitaxel, dexamethasone, avastin, cyclophosphamide, topotecan,olaparib, thiotepa, or a combination thereof.

In certain embodiments, a lyophilized formulation of Compound 1 providedherein is administered to patients with various types or stages ofprostate cancer, in combination with xeloda, 5 FU/LV, gemcitabine,irinotecan plus gemcitabine, cyclophosphamide, vincristine,dexamethasone, GM-CSF, celecoxib, taxotere, ganciclovir, paclitaxel,adriamycin, docetaxel, estramustine, Emcyt, denderon, zytiga,bicalutamide, cabazitaxel, degarelix, enzalutamide, zoladex, leuprolideacetate, mitoxantrone hydrochloride, prednisone, sipuleucel-T, radium223 dichloride, or a combination thereof.

In certain embodiments, a lyophilized formulation of Compound 1 providedherein is administered to patients with various types or stages of renalcell cancer, in combination with capecitabine, IFN, tamoxifen, IL-2,GM-CSF, Celebrex®, or a combination thereof.

In certain embodiments, a lyophilized formulation of Compound 1 providedherein is administered to patients with various types or stages ofgynecologic, uterus or soft tissue sarcoma cancer in combination withIFN, dactinomycin, doxorubicin, imatinib mesylate, pazopanib,hydrochloride, trabectedin, a COX-2 inhibitor such as Celebrex®, and/orsulindac.

In certain embodiments, a lyophilized formulation of Compound 1 providedherein is administered to patients with various types or stages of solidtumors in combination with celebrex, etoposide, cyclophosphamide,docetaxel, apecitabine, IFN, tamoxifen, IL-2, GM-CSF, or a combinationthereof.

In certain embodiments, a lyophilized formulation of Compound 1 providedherein is administered to patients with scleroderma or cutaneousvasculitis in combination with celebrex, etoposide, cyclophosphamide,docetaxel, apecitabine, IFN, tamoxifen, IL-2, GM-CSF, or a combinationthereof.

In certain embodiments, a lyophilized formulation of Compound 1 providedherein is administered to patients with MDS in combination withazacitidine, cytarabine, daunorubicin, decitabine, idarubicin,lenalidomide or a combination thereof.

Also encompassed herein is a method of increasing the dosage of ananti-cancer drug or agent that can be safely and effectivelyadministered to a patient, which comprises administering to the patient(e.g., a human) a lyophilized formulation of Compound 1 provided herein.Patients that can benefit by this method are those likely to suffer froman adverse effect associated with anti-cancer drugs for treating aspecific cancer of the skin, subcutaneous tissue, lymph nodes, brain,lung, liver, bone, intestine, colon, heart, pancreas, adrenal, kidney,prostate, breast, colorectal, or combinations thereof. Theadministration of a lyophilized formulation of Compound 1 providedherein alleviates or reduces adverse effects which are of such severitythat it would otherwise limit the amount of anti-cancer drug.

In one embodiment, a lyophilized formulation of Compound 1 providedherein is administered daily in an amount ranging from about 0.1 toabout 150 mg, from about 1 to about 50 mg, from about 2 to about 25 mg,or from about 1 to about 10 mg prior to, during, or after the occurrenceof the adverse effect associated with the administration of ananti-cancer drug to a patient. In certain embodiments, a lyophilizedformulation of Compound 1 provided herein is administered in combinationwith specific agents such as heparin, aspirin, coumadin, or G-CSF toavoid adverse effects that are associated with anti-cancer drugs such asbut not limited to neutropenia or thrombocytopenia.

In one embodiment, a lyophilized formulation of Compound 1 providedherein is administered to patients with diseases and disordersassociated with or characterized by, undesired angiogenesis incombination with additional active ingredients, including, but notlimited to, anti-cancer drugs, anti-inflammatories, antihistamines,antibiotics, and steroids.

In another embodiment, encompassed herein is a method of treating,preventing and/or managing cancer, which comprises administering alyophilized formulation of Compound 1 provided herein in conjunctionwith (e.g. before, during, or after) conventional therapy including, butnot limited to, surgery, immunotherapy, biological therapy, radiationtherapy, or other non-drug based therapy presently used to treat,prevent and/or manage cancer. The combined use of the compound providedherein and conventional therapy may provide a unique treatment regimenthat is unexpectedly effective in certain patients. Without beinglimited by theory, it is believed that a lyophilized formulation ofCompound 1 provided herein may provide additive or synergistic effectswhen given concurrently with conventional therapy.

As discussed elsewhere herein, encompassed herein is a method ofreducing, treating and/or preventing adverse or undesired effectsassociated with conventional therapy including, but not limited to,surgery, chemotherapy, radiation therapy, hormonal therapy, biologicaltherapy and immunotherapy. A lyophilized formulation of Compound 1provided herein and other active ingredient can be administered to apatient prior to, during, or after the occurrence of the adverse effectassociated with conventional therapy.

In certain embodiments, the methods provided herein compriseadministration of calcium, calcitriol, and vitamin D supplementationwith a lyophilized formulation of Compound 1. In certain embodiments,the methods provided herein comprise administration of calcium,calcitriol, and vitamin D supplementation prior to the treatment with alyophilized formulation of Compound 1.

In certain embodiments, calcium supplementation is administered todeliver at least 1200 mg of elemental calcium per day given in divideddoses. In certain embodiments, calcium supplementation is administeredas calcium carbonate in a dose of 500 mg administered three times a dayper orally (PO).

In certain embodiments, calcitriol supplementation is administered todeliver 0.25 μg calcitriol (PO) once daily.

In certain embodiments, vitamin D supplementation is administered todeliver about 500 IU to about 5000 IU vitamin D once daily. In certainembodiments, vitamin D supplementation is administered to deliver about1000 IU vitamin D once daily. In certain embodiments, vitamin Dsupplementation is administered to deliver about 50,000 IU vitamin Dweekly. In certain embodiments, vitamin D supplementation isadministered to deliver about 1000 IU vitamin D2 or D3 once daily. Incertain embodiments, vitamin D supplementation is administered todeliver about 50,000 IU vitamin D2 or D3 weekly.

In certain embodiments, a lyophilized formulation of Compound 1 providedherein and doxetaxol are administered to patients with non-small celllung cancer who were previously treated with carbo/VP 16 andradiotherapy.

6.6.2 Use With Transplantation Therapy

The lyophilized formulation of Compound 1 provided herein providedherein can be used to reduce the risk of Graft Versus Host Disease(GVHD). Therefore, encompassed herein is a method of treating,preventing and/or managing cancer, which comprises administering alyophilized formulation of Compound 1 provided herein in conjunctionwith transplantation therapy.

As those of ordinary skill in the art are aware, the treatment of canceris often based on the stages and mechanism of the disease. For example,as inevitable leukemic transformation develops in certain stages ofcancer, transplantation of peripheral blood stem cells, hematopoieticstem cell preparation or bone marrow may be necessary. The combined useof a lyophilized formulation of Compound 1 provided herein providedherein and transplantation therapy provides a unique and unexpectedsynergism. In particular, a lyophilized formulation of Compound 1provided herein exhibits immunomodulatory activity that may provideadditive or synergistic effects when given concurrently withtransplantation therapy in patients with cancer.

The lyophilized formulation of Compound 1 provided herein can work incombination with transplantation therapy reducing complicationsassociated with the invasive procedure of transplantation and risk ofGVHD. Encompassed herein is a method of treating, preventing and/ormanaging cancer which comprises administering to a patient (e.g., ahuman) lyophilized formulation of Compound 1 provided herein before,during, or after the transplantation of umbilical cord blood, placentalblood, peripheral blood stem cell, hematopoietic stem cell preparation,or bone marrow. Some examples of stem cells suitable for use in themethods provided herein are disclosed in U.S. Pat. No. 7,498,171, thedisclosure of which is incorporated herein by reference in its entirety.

In one embodiment, a lyophilized formulation of Compound 1 providedherein is administered to patients with acute myeloid leukemia before,during, or after transplantation.

In one embodiment, a lyophilized formulation of Compound 1 providedherein is administered to patients with multiple myeloma before, during,or after the transplantation of autologous peripheral blood progenitorcell.

In one embodiment, a lyophilized formulation of Compound 1 providedherein is administered to patients with NHL (e.g., DLBCL) before,during, or after the transplantation of autologous peripheral bloodprogenitor cell.

6.6.3 Cycling Therapy

In certain embodiments, the prophylactic or therapeutic agents providedherein are cyclically administered to a patient. Cycling therapyinvolves the administration of an active agent for a period of time,followed by a rest for a period of time, and repeating this sequentialadministration. Cycling therapy can reduce the development of resistanceto one or more of the therapies, avoid, or reduce the side effects ofone of the therapies, and/or improves the efficacy of the treatment.

Consequently, in certain embodiments, a lyophilized formulation ofCompound 1 provided herein provided herein is administered daily in asingle or divided dose in a four to six week cycle with a rest period ofabout a week or two weeks. In certain embodiments, a lyophilizedformulation of Compound 1 provided herein provided herein isadministered daily in a single or divided doses for one to tenconsecutive days of a 28 day cycle, then a rest period with noadministration for rest of the 28 day cycle. The cycling method furtherallows the frequency, number, and length of dosing cycles to beincreased. Thus, encompassed herein in certain embodiments is theadministration of a lyophilized formulation of Compound 1 providedherein for more cycles than are typical when it is administered alone.In certain embodiments, a lyophilized formulation of Compound 1 providedherein is administered for a greater number of cycles that wouldtypically cause dose-limiting toxicity in a patient to whom a secondactive ingredient is not also being administered.

In one embodiment, a lyophilized formulation of Compound 1 providedherein is administered daily and continuously for three or four weeks ata dose of from about 0.1 to about 150 mg/d followed by a break of one ortwo weeks.

In another embodiment, a lyophilized formulation of Compound 1 providedherein is administered intravenously and a second active ingredient isadministered orally, with administration of the lyophilized formulationof Compound 1 occurring 30 to 60 minutes prior to a second activeingredient, during a cycle of four to six weeks. In certain embodiments,the combination of a lyophilized formulation of Compound 1 providedherein and a second active ingredient is administered by intravenousinfusion over about 90 minutes every cycle. In certain embodiments, onecycle comprises the administration from about 0.1 to about 150 mg/day ofa lyophilized formulation of Compound 1 provided herein and from about50 to about 200 mg/m²/day of a second active ingredient daily for threeto four weeks and then one or two weeks of rest. In certain embodiments,the number of cycles during which the combinatorial treatment isadministered to a patient is ranging from about one to about 24 cycles,from about two to about 16 cycles, or from about four to about threecycles.

6.6.4 Patient Population

In certain embodiments of the methods provided herein, the subject is ananimal, preferably a mammal, more preferably a non-human primate. Inparticular embodiments, the subject is a human. The subject can be amale or female subject.

Particularly useful subjects for the methods provided herein includehuman cancer patients, for example, those who have been diagnosed withleukemia, including acute myeloid leukemia, acute lymphocytic leukemia,chronic myelogenous leukemia, and chronic myelogenous leukemia. Incertain embodiments, the subject has not been diagnosed with acutepromyelocytic leukemia.

In some embodiments, the subject has a higher than normal blastpopulation. In some embodiments, the subject has a blast population ofat least 10%. In some embodiments, the subject has a blast population ofbetween 10 and 15%. In some embodiments, the subject has a blastpopulation of at least 15%. In some embodiments, the subject has a blastpopulation of between 15 and 20%. In some embodiments, the subject has ablast population of at least 20%. In some embodiments, the subject has ablast population of about 10-15%, about 15-20%, or about 20-25%. Inother embodiments, the subject has a blast population of less than 10%.In the context of the methods described herein, useful subjects having ablast population of less than 10% includes those subjects that, for anyreason according to the judgment of the skilled practitioner in the art,are in need of treatment with a compound provided herein, alone or incombination with a second active agent.

In some embodiments, the subject is treated based on the EasternCooperative Oncology Group (ECOG) performance status score of thesubject for leukemia. ECOG performance status can be scored on a scaleof 0 to 5, with 0 denoting asymptomatic; 1 denoting symptomatic butcompletely ambulant; 2 denoting symptomatic and <50% in bed during theday; 3 denoting symptomatic and >50% in bed, but not bed bound; 4denoting bed bound; and 5 denoting death. In some embodiments, thesubject has an ECOG performance status score of 0 or 1. In someembodiments, the subject has an ECOG performance status score of 0. Insome embodiments, the subject has an ECOG performance status score of 1.In other embodiments, the subject has an ECOG performance status scoreof 2.

In certain embodiments, the methods provided herein encompass thetreatment of subjects who have not been previously treated for leukemia.In some embodiments, the subject has not undergone allogeneic bonemarrow transplantation. In some embodiments, the subject has notundergone a stem cell transplantation. In some embodiments, the subjecthas not received hydroxyurea treatment. In some embodiments, the subjecthas not been treated with any investigational products for leukemia. Insome embodiments, the subject has not been treated with systemicglucocorticoids.

In other embodiments, the methods encompass treating subjects who havebeen previously treated or are currently being treated for leukemia. Forexample, the subject may have been previously treated or are currentlybeing treated with a standard treatment regimen for leukemia. Thesubject may have been treated with any standard leukemia treatmentregimen known to the practitioner of skill in the art. In certainembodiments, the subject has been previously treated with at least oneinduction/reinduction or consolidation AML regimen. In some embodiments,the subject has undergone autologous bone marrow transplantation or stemcell transplantation as part of a consolidation regimen. In someembodiments, the bone marrow or stem cell transplantation occurred atleast 3 months prior to treatment according to the methods providedherein. In some embodiments, the subject has undergone hydroxyureatreatment. In some embodiments, the hydroxyurea treatment occurred nolater than 24 hours prior to treatment according to the methods providedherein. In some embodiments, the subject has undergone prior inductionor consolidation therapy with cytarabine (Ara-C). In some embodiments,the subject has undergone treatment with systemic glucocorticosteroids.In some embodiments, the glucocorticosteroid treatment occurred no later24 hours prior to treatment according to the methods described herein.In other embodiments, the methods encompass treating subjects who havebeen previously treated for cancer, but are non-responsive to standardtherapies.

Also encompassed are methods of treating subjects having relapsed orrefractory leukemia. In some embodiments, the subject has been diagnosedwith a relapsed or refractory AML subtype, as defined by the WorldHealth Organization (WHO). Relapsed or refractory disease may be de novoAML or secondary AML, e.g., therapy-related AML (t-AML).

In some embodiments, the methods provided herein are used to treat drugresistant leukemias, such as chronic myelogenous leukemia (CML). Thus,treatment with a compound provided herein could provide an alternativefor patients who do not respond to other methods of treatment. In someembodiments, such other methods of treatment encompass treatment withGleevec® (imatinib mesylate). In some embodiments, provided herein aremethods of treatment of Philadelphia chromosome positive chronicmyelogenous leukemia (Ph+CML). In some embodiments, provided herein aremethods of treatment of Gleevec® (imatinib mesylate) resistantPhiladelphia chromosome positive chronic myelogenous leukemia (Ph+CML).

Also encompassed are methods of treating a subject regardless of thesubject's age, although some diseases or disorders are more common incertain age groups. In some embodiments, the subject is at least 18years old. In some embodiments, the subject is more than 18, 25, 35, 40,45, 50, 55, 60, 65, or 70 years old. In other embodiments, the subjectis less than 65 years old. In some embodiments, the subject is less than18 years old. In some embodiments, the subject is less than 18, 15, 12,10, 9, 8 or 7 years old.

In some embodiments, the methods may find use in subjects at least 50years of age, although younger subjects could benefit from the method aswell. In other embodiments, the subjects are at least 55, at least 60,at least 65, and at least 70 years of age. In another embodiment, thesubjects have adverse cytogenetics. “Adverse cytogenetics” is defined asany nondiploid karyotype, or greater than or equal to 3 chromosomalabnormalities. In another embodiment, the subjects are at least 60 yearsof age and have adverse cytogenetics. In another embodiment, thesubjects are 60-65 years of age and have adverse cytogenetics. Inanother embodiment, the subjects are 65-70 years of age and have adversecytogenetics.

In certain embodiments, the subject treated has no history of myocardialinfarction within three months of treatment according to the methodsprovided herein. In some embodiments, the subject has no history ofcerebrovascular accident or transient ischemic attack within threemonths of treatment according to the methods provided herein. In someembodiments, the subject has no suffered no thromboembelic event,including deep vein thrombosis or pulmonary embolus, within 28 days oftreatment according to the methods provided herein. In otherembodiments, the subject has not experienced or is not experiencinguncontrolled disseminated intravascular coagulation.

Because subjects with cancer have heterogeneous clinical manifestationsand varying clinical outcomes, the treatment given to a patient mayvary, depending on his/her prognosis. The skilled clinician will be ableto readily determine without undue experimentation specific secondaryagents, types of surgery, and types of non-drug based standard therapythat can be effectively used to treat an individual subject with cancer.

It will be appreciated that every suitable combination of the compoundsprovided herein with one or more of the aforementioned compounds andoptionally one or more further pharmacologically active substances iscontemplated herein.

6.7 Evaluation of Activity

Standard physiological, pharmacological and biochemical procedures areavailable for testing the compounds to identify those that possess thedesired anti-proliferative activity.

Such assays include, for example, biochemical assays such as bindingassays, radioactivity incorporation assays, as well as a variety of cellbased assays, including KG-1 cell proliferation assay described in theExample section.

Embodiments provided herein may be more fully understood by reference tothe following examples. These examples are meant to be illustrative ofpharmaceutical compositions and dosage forms provided herein, but arenot in any way limiting.

7. EXAMPLES

The following Examples are presented by way of illustration, notlimitation. The following abbreviations are used in descriptions andexamples.

SWFI—Sterile Water for Injection

WFI—Water for injection

D5W—Dextrose 5% in Water

HPβCD—Hydroxypropyl-beta-cyclodextrin

SBEβCD—Sulfobutylether-β-cyclodextrin sodium salt

TBA—Tert-butyl alcohol

DMA—Dimethylacetamide

HAS—Human serum albumin

FDM—Freeze-drying microscope

SEM—Scanning electron microscope

LT-DSC—Low temperature differential scanning calorimetry

DSC—Differential scanning calorimetry

DVS Dynamic vapor sorption

TGA—Thermogravimetic analysis

GC—Gas chromatography

KF—Karl Fisher

“Compound 1, Form C” or “Form C” or “API”” in the Examples herein refersto polymorph Form C of2-(4-Chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamide.“Compound 1, Form A” or “Form A” in the Examples herein refers topolymorph Form A of2-(4-Chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamide.The physical and chemical properties of2-(4-Chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamideare summarized in Table 1.

TABLE 1 Summary of physical and chemical properties of2-(4-Chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamideStructure

Molecular C₂₂H₁₈ClF₂N₃O₄ Formula Molecular 461.85 Weight Log D cLogP =2.18 (Log D not measured due to solubility) pKa cpKa = 10.66 (Notmeasured due to low stability above pH 7) Melting Point 234° C. (Form C)Appearance White powder Solubility Practically insoluble in water (≤1μg/ml across pH range of 1-8) Solid State DS is physically stable underall storage conditions. Stability Solution Stability DS is not stable insolution at pH of 5.0 or above. Hydrolysis is the major degradationpathway. Hygroscopicity Not hygroscopic Pharmaceutical Crystalline;Anhydrous; five polymorph forms (Form C being the Form most stable form)

Example 1: First Formulation Screen

Due to the poor water solubility of Compound 1, Form C, a cosolventsystem was required to solubilize the drug compound in a solution priorto lyophilization. Several solubilizing agents and solvents that wereconsidered in this example include hydroxypropyl-beta-cyclodextrin(HPβCD), Sulfobutylether-β-cyclodextrin sodium salt (SBEβCD; Captisol®),Tert-butyl alcohol (TBA), and dimethylacetamide (DMA). The solubilitiesof Compound 1, Form A in various cosolvent systems were determined andthe results are shown in Table 2.

TABLE 2 Form C Form A Vehicle (mg/ml) (mg/ml) 5% HP-β-CD NT 0.03 10%HP-β-CD NT 0.06 20% HP-β-CD NT 0.15 5% Captisol ® NT 0.03 10% Captisol ®NT 0.06 20% Captisol ® 0.10 0.13 30% Captisol ® 0.19 0.30 40% Captisol ®NT 0.32 TBA NT 0.06 DMA <166, >125 NT 20:80 TBA/pH 4.5 buffer NT 0.0140:60 TBA/pH 4.5 buffer NT 0.19 60:40 TBA/pH 4.5 buffer 0.29 0.39 70:30TBA/pH 4.5 buffer 0.31 0.42 80:20 TBA/pH 4.5 buffer 0.33 0.47 90:10TBA/pH 4.5 buffer 0.23 0.35 80% TBA/15% pH 4.5 buffer/5% DMA 0.47 NT 80%TBA/10% pH 4.5 buffer/10% DMA 0.58 NT 70% TBA/25% pH 4.5 buffer/5% DMA0.47 NT 70% TBA/20% pH 4.5 buffer/10% DMA 0.66 NT

It was found that HPβCD and SBEβCD had comparable solubilizing effectson Form A at a concentration range of 5-20%. When the SBEβCDconcentration increased from 30% to 40%, the enhancement in drugsolubility was very limited. It was also found that the drug solubilityreached the highest when the TBA/water ratio was at 80:20. Thesolubility measurements of Form C in selected cosolvent systems wereconducted and the results are also shown in Table 2.

In the first formulation screen, the focus was on various combinationsof solvents and excipients that provided a media which allowed adequatedrug solubility and stability throughout the process of lyophilizationand storage of the final product. The 20 mM citrate buffer solution atpH 4.5 was used as the aqueous phase noting that the drug compoundundergoes hydrolysis degradation in solutions at pH 5 or above. Given atarget drug loading of 2 mg/vial and the maximum fill volume of 8 ml ina 20 cc vial container, the minimum feasible drug concentration in thebulk solution was 0.25 mg/ml. As shown in Table 2, The API has thehighest solubility of 0.33 mg/ml in 80:20 TBA/pH4.5 buffer. However, thesolubilities of many commonly used bulking agents such as mannitol,sucrose, and glycine are significantly limited in the 70% v/v or higherTBA solution. To balance the solubility of both API and the excipientsin the bulk solution, a 60:40 TBA/pH4.5 citrate buffer solution was usedin the first screen as the starting point.

In the first run of formulation screen, seven prototype formulationswere prepared with different excipients: mannitol, sucrose, PlasdoneC17, Captisol®, proline and glycine.

Table 3 shows the formulations compositions of the seven sublots in thefirst screen.

TABLE 3 Formulation No. I II III IV V VI VII API (mg/ml) 0.24 0.24 0.280.24 0.24 0.24 0.26 Mannitol (mg/ml) 21 25 21 Plasdone C17 (mg/ml) 17Sucrose (mg/ml) 25 Glycine (mg/ml) 17 19 PEG300 (mg/ml) 4 Captisol ®(mg/ml) 17 Proline (mg/ml) 7 TBA (% v/v) 60 68.6 55.7 58.1 60 59.6 56.920 mM citric buffer 40 31.4 39.3 40 (% v/v) DMA (% v/v) 4.9 1.6 Purifiedwater (% v/v) 39.7 39.7 39 0.1N HCl (% v/v) 2.2 0.3 2.5 0.1N NaOH (%v/v) 0.4

In Formulations I, II and V, the excipients were dissolved in 20 mMcitric buffer solution (pH=4.7) first, then mixed with TBA. SubsequentlyAPI was dissolved in the TBA/buffer mixture. In Formulations IV and VI,the excipients were dissolved in purified water followed by a pHadjustment with HCl and the addition of TBA. Then the drug was directlydissolved in the TBA/water mixture. In Formulations III and VII, the APIwas dissolved in a small amount of DMA first, and then added to theTBA/buffer or TBA/water solution. It was observed that the solutionbecame hazy after the addition of TBA in Formulations IV and VII. It wassuspected that glycine in those formulations may have reached itssolubility limit in the TBA/water solution.

The pH values of the bulk solution in each preparation step weremeasured and reported in Table 4.

TABLE 4 Sublot (Formulation Nos.) I II III IV V VI VII pH of 4.8 4.8 4.76.2 4.8 5.0 6.2 excipients in adjusted adjusted adjusted Water or to 4.5to 4.5 to 4.5 Buffer pH of 5.7 6.1 5.6 5.0 5.8 5.2 4.9 excipients postTBA addition pH post API 5.8 6.1 5.9 5.0 5.8 5.2 5.0 addition Final pH(pre 5.8 6.1 5.9 5.0 5.8 5.2 5.0 filtration) Final pH (post 5.8 6.1 5.95.0 5.6 5.3 5.1 filtration) Post 4.9 5.0 5.0 4.7 5.0 7.7 4.6reconstitution

API addition and filtration had no impact on the pH of the solution. Theaddition of TBA to the solution caused a big increase in the pH reading,which may not reflect the true pH of the solution because the presenceof the organic solvent often interferes with the measurement of the pHmeter probes. The pH values of the solutions post reconstitution withpurified water were all kept at 5.0 or less except for Formulation VI. Ageneric and conservative lyophilization cycle was applied to all theseven screened formulations and the process parameters of each freezedrying step were described in Table 5.

TABLE 5 Shelf Temp. Ramping Setpoint Soak Time Rate Step (° C.) (hours)(° C./hour) Pressure Setpoint Product 5 2 30 Evac. To 12 psia Loading/to ensure Freezing chamber is Freezing −50 3 30 airtight Primary −28 2130 60 microns Drying Secondary 25 6 Drying Stoppering 25 14.7 PSIA

All the seven formulations displayed acceptable cake appearances afterlyophilization. The assay and purity of the lyophilized cakes weremeasured by HPLC and the moisture content of each formulation wasmeasured by Karl Fisher. The results are shown in Table 6.

TABLE 6 Sublot (Formulation Nos.) I II III IV V VI VII Purity (% area)99.3 71.7 99.3 99.2 99.4 99.2 84.6 Assay (% LC) 95.4 93.9 95.9 112.1108.3 106.5 78.3 Moisture −0.09 0.30 0.04 1.27 0.03 0.16 0.21 content (%w/w)

Formulation VII was ruled out due to its low assay and purity,indicating that glycine and proline may not be adequate stabilizers inthe lyophilization process. Formulation II showed low purity due to manyinterfering peaks of the polymeric excipient in the HPLC chromotograph.It remained to be investigated in the next run.

A few key observations concluded from the first screen are:

Adjusting pH with HCl and/or NaOH exhibited limited buffering capacityand introduced pH variation during the process. Using citrate bufferoffered more consistent and robust pH control.

Dissolving the drug substance first to DMA and then adding the API/DMApremix to the respective TBA/buffer solution aided in initialdissolution of the drug substance, which allowed for higher drugconcentration and lower TBA concentration in the final bulk solution.

Formulation VI, the formulation containing Captisol®, was able to bereconstituted by purified water or D5W alone at a concentration of0.24-0.5 mg/ml. All the other formulations required some cosolvents suchas ethanol or PEG300 for complete reconstitution.

Example 2: Second Formulation Screen

The preliminary results from the first screen confirmed thelyophilization feasibility of the drug compound. The second formulationscreen was designed to evaluate the physical and chemical stability ofmultiple prototype formulations and to select the lead formulationcandidates. The same generic lyophilization cycle as used in the firstscreen was adopted. 20 mM citrate buffer at pH 4.5 was used in all theformulations except for the one containing glycine (Formulation XIV).API was dissolved in DMA first at a concentration of 40 mg/ml. Then theAPI/DMA premix was added to the TBA/buffer solution containing theexcipients. Dexolve and Kleptose® HPB are the two derivatives ofcyclodextrins which have similar physical and chemical properties asCaptisol®. They were included in Formulations VIII, IX and X as thealternatives of Captisol®. Mannitol, Plasdone, sucrose and glycineexhibited acceptable formulation characteristics in the first screen andthus continued to be evaluated in the second screen. The excipientlevels were adjusted empirically in order to obtain a clear andcolorless bulk solution. Formulation XI, the formulation containinghuman serum albumin (HSA), was discarded during the preparation due toincomplete dissolution of the API. The formulation compositions of theseven sublots are shown in Table 7.

TABLE 7 Formulation No. VIII IX X XII XIII XIV XV API (mg/ml) 0.28 0.240.3 0.3 0.3  0.28 0.28 Mannitol (mg/ml) 12 Plasdone C17 (mg/ml) 13.3Sucrose (mg/ml) 13.3 12   Glycine (mg/ml) 6.1 PEG300 (mg/ml) 6.6 Dexolve(mg/ml) 24.5 Kleptose ® (mg/ml) 21.5 13.3 TBA (% v/v) 50.4 56.6 46.346.3 46.3 50.4  50.4 20 mM citric buffer 49 42.9 53 53 53 49 (% v/v) DMA(% v/v) 0.7 0.6 0.7 0.7 0.7 0.7 0.7 Purified water (% v/v) 49*  

The fill volume of each vial was about 2.5-3 ml to deliver a drugloading of 0.75-0.77 mg/vial. The total solid content of the lyophilizedcakes ranged from 40 mg to 75 mg per vial. As shown in Table 8, the pHvalues of the bulk solutions post reconstitution were all kept around4.5, which confirmed the buffer capacity of the formulation.

TABLE 8 Sublot (Formulation No.) VIII IX X XII XIII XIV XV pH of 4.6 4.74.7 4.8 4.8 4.6 4.7 excipients in Water or Buffer plus TBA pH post API5.2 5.5 5.2 5.2 5.3 4.7 5.3 and TBA addition Final pH (post 5.1 5.6 5.45.4 5.4 4.9 5.3 filtration) pH Post 4.5 4.6 4.5 4.5 4.5 4.4 4.5reconstitution

The physical structures of the seven formulations after lyophilizationwere fully characterized and the results are shown in Table 9.

TABLE 9 Formulation VIII IX X XII Color White, Uniform White, UniformWhite, Uniform White, Uniform Structure Dense Dense Porous Powder FillHeight 9 mm 8 mm 5 mm 5 mm Cake Height 4 mm 6 mm 3 mm n/a Side 2 mmuniform 1 mm uniform 2 mm n/a Shrinkage Top Surface Matte SheenMatte/Sheen n/a Side Surface Matte Matte Matte n/a Topography Textured,concave Textured, Textured, concave n/a concave Upon Cake remains intactCake remains Cake remains mostly n/a Inversion and falls to top ofintact and adhered intact with some vial. to bottom of vial. chunksdislodged. Upon Cake fractures into Cake falls to the Cake fracturesinto n/a Jarring pieces. top of vial and chunks. powder. remains intactResidual Minimal; bottom Minimal; bottom Minimal Minimal Material withstriations with striations

Formulations VIII, XIX and XV exhibited elegant cake appearances whileFormulations X, XII, XIII and XIV did not retain a good cake structure,with collapsed or fractured chunks of powders in the vial.

All the lyophilized samples were put on stability at 25° C./60% RH and40° C./75% RH condition. The assay and purity data of each formulationare shown in Table 10. The results indicated that all the formulationsin the second screen remained chemically stable for at least 1 month atthe long-term and the accelerated stability condition.

TABLE 10 Formulation VIII IX X XII XIII XIV XV Initial Purity 97.8 98.599.5 98.7 98.2 98.5 99.0 (% area) Assay 93.5 92.1 90.7 92.0 95.2 96.1103.9 (% LC) 40° C./ Purity 97.8 98.6 99.3 98.8 98.4 98.7 99.2 75% RH (%area 1 wk Assay 93.5 93.4 92.0 93.3 97.3 100.0 105.2 (% LC) 40° C./Purity 97.9 98.5 99.3 98.8 98.2 98.7 99.1 75% RH (% area 2 wk Assay 96.196.1 93.3 96.0 93.3 93.5 102.6 (% LC) 40° C./ Purity 97.8 98.6 99.1 98.998.4 98.9 99.2 75% RH (% area) 4 wk Assay 97.4 96.1 97.3 96.0 94.7 90.9106.5 (% LC) 25° C./ Purity 97.9 98.5 99.3 98.8 98.2 98.6 99.1 60% RH (%area) 2 wk Assay 96.1 96.1 93.3 96.0 90.7 97.4 102.6 (% LC) 25° C./Purity 97.8 98.6 99.4 98.8 98.2 98.7 99.2 60% RH (% area) 4 wk Assay97.4 96.1 93.3 96.0 93.3 101.3 106.5 (% LC)

The physical stability of the seven formulations were characterized byXRPD. The results are illustrated in FIGS. 30A, 30B and 30C. For theinitial samples, all the formulations exhibited amorphous forms exceptfor Formulation XIV and XV. A further scan verified that Formulation XIVpeaks corresponded to the β-form of glycine and Formulation XV peakscorresponded to the mixed α-form and δ-form of mannitol. After thestability samples were stored at 25° C./60% RH for 1 month, a few smallpeaks of sucrose showed up in the XRPD profile of Formulation XIV inaddition to the glycine peaks, and the sucrose peaks also showed up inthe XRPD patterns of the Formulation XIII sample. The similar changes inXRPD patterns were observed in the Formulations XIII and XIV 1-monthstability samples at 40° C./75% RH to a greater extent. In the meantime, a couple of Formulations XIII and XIV samples after one monthstorage at 40° C./75% RH were found to exhibit slight color change fromoff-white to yellowish and the lyophilized powders became tacky.

The reconstitution study was executed on each formulation with fourdifferent diluents, namely purified water, D5W, 50% v/v ethanolsolution, and 50% v/v PEG300 in D5W solution. The shaking time requiredfor the complete dissolution and the physical appearance of thereconstituted solution were inspected. The results are shown in Table11.

TABLE 11 50:50 50:50 Purified Water D5W Ethanol/water PEG300/D5W Sublot(3 mL) (2 ml) (4 ml) (2 ml) Formulation 20 s 20 s 30 s, c/c 30 s, c/cVIII c/c; became hazy c/c; became hazy after 2 h after 2 h Formulation40 s 40 s 30 s, c/c 30 s, c/c IX c/c; became hazy c/c; became hazy after2 h after 2 h Formulation X 40 s 40 s 30 s, c/c 30 s, c/c c/c; becamehazy c/c; became hazy after 2 h after 2 h Formulation >25 m >25 m 30 s,c/c 30 s, c/c XII Slightly cloudy Slightly cloudy Formulation >25 m >25m 30 s, c/c 30 s, c/c XIII Slightly cloudy Slightly cloudyFormulation >25 m >25 m 30 s, c/c 30 s, c/c XIV Slightly cloudy Slightlycloudy Formulation >25 m >25 m 30 s, c/c 30 s, c/c XV Slightly cloudySlightly cloudy

Formulation VIII, the dexolve-based formulation, was able to bereconstituted to a clean and colorless solution with 2-3 ml purifiedwater or D5W alone within 20 seconds. Formulations IX and X, theKleptose®-based formulations, were able to be reconstituted with 2-3 mlpurified water or D5W alone as well, but the complete reconstitutionrequired 40-60 seconds. The reconstituted solutions of all the threeformulations became hazy after 2 hours, implying that a larger volume ofreconstitution diluent may be required to achieve longer in-usestability before the drug is precipitated out of the solution. The restof the four formulations were not able to be reconstituted by the samevolume of the purified water or D5W alone. When 50:50 ethanol/watersolution or 50:50 PEG300/D5W solution was used as the reconstitutiondiluent, all the seven formulations were able to be reconstituted with 2ml of the diluent within 30 seconds. As the solvent-free diluent is mostpreferable in terms of easiness of formulation preparation and excipienttolerability, the Dexolve or Kleptose® based formulations areadvantageous over the other formulation candidates.

The moisture content of a lyophilized sample could have great impact onits physical and chemical stability. The water content of eachlyophilized sample was measured by Karl Fisher. As shown in Table 12,the water contents of all the formulations were less than 0.5% exceptfor Formulation XIV which had a water content of 1.25%.

TABLE 12 TGA First (Lowest) Formulation KF Residual Water Weight Lossthermal event Nos. (% w/w) (% w/w) (peak ° C.) Formulation 0.50 3.07(loss 1) 120.8  VIII 4.21 (loss 2) 7.28 (Total) Formulation 0.04 11.4 93.7 IX Formulation X −0.09 2.79 86.5 Formulation 0.08 6.04 80.8 XIIFormulation −0.17 2.98 n/d XIII Formulation 1.25 6.74 47.3 XIVFormulation 0.14 3.07 (loss 1) (46.8 exo) XV 2.02 (loss 2) 179.1  5.09(Total)

The total weight loss of each lyophilized sample upon heating from roomtemperature up to 200° C. was obtained from the TGA measurement. Giventhe low water content of each sample as indicated in the Karl Fishermeasurement, the majority of the weight loss was attributed to theresidual solvent held in the lyophilized cake. It showed in Table 12that the weight loss across all the formulations ranged from 2.8% to11.4%, implying that most of the lyophilized samples carried relativelyhigh residual solvents. Residual solvent level of the final drugproducts may cause serious toxicity concerns and therefore need to betightly controlled according to the ICH guideline Q3C. A GC method wasdeveloped upon this point for the quantification of the residual solventlevel of the lyophilized formulations for the further development work.The DSC measurement was also conducted on each sample to characterizethe thermal response of the lyophilized material. The lowest peaktemperature of the first endothermic event identified from the DSCprofile reflects the glass transition temperature of the amorphousmaterial. Formulations VIII, IX and XV showed relatively higher glasstransition temperatures than the other formulations. In correspondence,these three formulations also exhibited better cake appearance andintegrity than the others. Formulations XIII and XIV had the lowestglass transition temperatures among the seven screened formulations,which partially explained their weak physical stability observed at theaccelerated stability condition.

Example 3: Third Formulation Screen

As the cake integrities of several formulations in the second screenwere not as good as expected, the bulking agent level in theformulations was increased to help stabilize the cake structure duringthe lyophilization cycle. In order for the bulking agents to achieve aconcentration of 50 mg/ml, it was found that the TBA concentration inthe bulk solution should be kept at 55% v/v or less. The new lot of APImaterial was dissolved in DMA at a concentration of 60 mg/ml and thenadded to 50% TBA in citrate buffer solution. The drug concentration inthe final bulk solution may increase from previous 0.3 mg/ml up to 1mg/ml with the increased ratio of API/DMA concentrate. In the thirdscreen, in order to achieve a target drug loading of 2 mg/vial, the drugconcentration in the final bulk solution was set at 0.5 mg/ml,corresponding to 4 ml of the fill volume in the vial. FormulationsXVI-XXII carried on the same bulking agents as used in the previousscreen but at a higher bulking agent level of 50 mg/ml. In FormulationXXIII, a combination of Captisol® and Plasdone C17 was used as bulkingagents to test if the addition of the polymer would enhance thesolubility of the drug compound so that the same drug concentration canbe achieved at a lower TBA level of 30% v/v instead of 50% v/v. Table 13shows the formulation compositions of the bulk solutions in the thirdscreen.

TABLE 13 Formulation No. XVI XVII XVIII XIX XX XXI XXII* XXIII API(mg/ml) 0.5  Mannitol (mg/ml) 50 Plasdone C17 (mg/ml) 50 5 Sucrose(mg/ml) 50 35 Glycine (mg/ml) 15 Captisol ®(mg/ml) 50 45 Dexolve (mg/ml)50 Kleptose ® (mg/ml) 50 TBA (% v/v) 49.58 29.75 Citric buffer (% v/v)49.58 69.42 DMA (% v/v) 0.83 *pH adjusted by HCl instead of citricbuffer

The pH values of the final bulk solutions and the solution postreconstitution of all the screened formulations were kept well below5.0, as shown in Table 14.

TABLE 14 Formulation Formulation Formulation Formulation FormulationFormulation Formulation Formulation Sublot XVI XVII XVIII XIX XX XXIXXII XXIII pH of excipients in 4.6 4.7 5.0 5.0 5.0 5.0 4.3 4.5 50% v/vTBA/water Final pH including 4.9 4.9 5.2 5.2 5.1 5.1 4.4 4.7 API (postfiltration) Post reconstitution 4.3 4.3 4.2 4.3 4.1 4.3 3.9 4.3

The same generic lyophilization cycle as used in the first screen wasadopted in this study. The physical structures of the eight lyophilizedformulations were fully characterized and the results are shown in Table15.

TABLE 15 Formulation Formulation Formulation Formulation CharacteristicXVI XVII XVIII XIX Color White, White, Uniform White, Uniform White,Uniform Uniform Structure Dense Dense Dense Dense Fill Height 8 mm 8 mm8 mm 8 mm Cake Height 3-5 mm 4-5 mm 6-8 mm 3-6 mm Side Shrinkage 3 mmuniform 2 mm uniform 1 mm uniform 2 mm uniform Top Surface Matte MatteMatte w/ a sheen Matte and skin Sheen Side Surface Matte Matte Matte,with slight Matte, with creep up the sides slight creep up the sidesTopography Textured, Textured, Textured, concave Textured, concave atconcave at edges, at edges, ⅔ of concave at edges, multiple multiplepeaks surface has a sheen edges, surface is peaks skin slightly porousUpon Inversion Cake remains Cake remains Cake remains intact Cakeremains intact and falls intact and falls to and falls to top of intactand falls to top of vial. top of vial. vial. to top of vial. UponJarring Cake falls to Cake falls to top Cake falls to top of Cake fallsto top top of vial of vial and breaks vial and breaks into of vial SmallSmall pieces into pieces. pieces. pieces fall off. fall off. ResidualMinimal Minimal Minimal Minimal Material Formulation FormulationFormulation Characteristic XX XXI Formulation XXII XXIII Color White,White, Uniform White, Uniform White, Uniform Uniform Structure DenseDense Porous Dense Fill Height 8 mm 8 mm 8 mm 8 mm Cake Height 7-8 mm4-5 mm 3-4 mm 3-5 mm Side Shrinkage 2 mm uniform 2 mm uniform N/A 2 mmuniform Top Surface Matte and Matte and Sheen Matte Matte and SheenSheen Side Surface Matte, with Matte, with slight Matte Matte smallcreep up creep up the sides the sides. Topography Textured, Textured,Textured Smooth, concave at concave at edges, concave along edges,multiple multiple peaks the edges, ½ of peaks surface has a skin. UponInversion Cake remains Cake remains Cake remains intact Cake remainsintact and falls intact and falls to and falls to top of intact andfalls to top of vial. top of vial. vial. to top of vial. Upon JarringCake falls to Cake falls to top Cake falls to top of Cake falls to toptop of vial and of vial and breaks vial and powderizes of vial Smallpowderizes into pieces amount falls off. Residual Significant MinimalMinimal Minimal Material

With the increased level of the bulking agents, all the formulationsobtained acceptable cake appearance except for Formulation XXII, the onecontaining sucrose and glycine. The observation was consistent with whathad been seen in Formulation XIV which contained lower levels of sucroseand glycine. As a result of the poor cake integrity and the deterioratedphysical stability observed in the second screen, the formulationscontaining sucrose and/or glycine were ruled out.

The reconstitution performance of the eight formulations in the thirdscreen was consistent with that observed in the second screen.Formulations XVI, XVII, XVIII, the cyclodextrin-based formulations, arethe only three formulations that can be reconstituted by purified wateror D5W alone. All the rest formulations required cosolvent diluent forcomplete dissolution including Formulation XXIII Surprisingly, althoughit contained Captisol®, the drug solubility of Formulation XXIII waslimited rather than enhanced by the presence of Plasdone. ThereforeFormulation XXIII was abandoned from the screen.

The Karl Fisher, DSC, and TGA results were summarized in Table 16.

TABLE 16 TGA First (Lowest) DSC KF Residual Water Weight Loss ThermalEvent Sublot (% w/w), 2 vials (% w/w) (peak ° C.) Formulation 0.11, 0.03 2.42 (loss 1) 112.7 XVI  3.68 (loss 2)  6.10 (Total) Formulation 0.18,0.06  2.96 (loss 1) 126.5 XVII  5.05 (loss 2)  8.01 (Total) Formulation0.08, 0.00  1.61 (loss 1) 89.6 XVIII  9.29 (loss 2) 10.90 (Total)Formulation 0.11, 0.02  1.81 (loss 1) 95.0 XIX  6.60 (loss 2)  3.55(loss 3) 11.96 (Total) Formulation 0.15, 0.30  1.41 (loss 1) 98.5 XX 0.21 (loss 2)  1.62 (Total) Formulation 0.03, 0.05  1.82 (loss 1) 64.6XXI  0.78 (loss 2)  2.75 (loss 3)  2.46 (loss 4)  7.81 (Total)Formulation 0.23, 0.33 n/d 58.7 XXII Formulation 0.41, 0.28  2.09(loss 1) 105 XXIII  5.37 (loss 2)  7.46 (Total)

The water contents detected by the Karl Fisher measurements showed thatall the formulations contained less than 0.5% water. However, the TGAresults indicated that the weight loss of each formulation upon heatingvaried in a wide range, which predominantly resulted from a high levelof residual solvent in the lyophilized cakes. Formulations XVIII and XIXcontained relatively higher residual solvent levels than the otherformulations, plausibly due to the tendency of solvent molecules boundwith the sugar ring of the cyclodextrin and polymeric chain of thepovidone. Formulation XX, the mannitol-based formulation, contained thelowest residual solvent of 1.6%. Table 17 provides residual solvents ofthe selected formulations from the third screen.

TABLE 17 Formulation TBA (mg/vial) DMA (mg/vial) Formulation XVI(Dexolve) 1.4 11.0 Formulation XVII (Captisol ®) 9.0 19.1 FormulationXVIII 11.7 11.0 (Kleptose ®) Formulation XX (Mannitol) 0.12 3.18

With the completion of the third formulation screen, the Captisol® orDexolve-based formulation stood out as the lead candidate due to itsgood physical and chemical stability as well as its fast and simplereconstitution. The Kleptose®-based formulation was considered as thealternative as it displayed similar characteristics as theCaptisol®-based formulation. The mannitol-based formulation was alsoconsidered as the backup due to its superior cake structure and thelowest residual solvent level among all the screened formulations.

The GC tests were conducted on the aforementioned four lead formulationsto acquire a more definite amount of the residual solvents in eachindividual vial. Therefore, the next run of formulation screen wasfocused on reducing the solvent level used in the initial bulk solution.Dexolve was used as the sole bulking agent in the screen provided thedrug has comparable solubility in Captisol®, Dexolve, or Kleptose®solutions.

Example 4: Fourth Formulation Screen

All the previous formulation development work was targeted at a drugloading of 2 mg/vial. A drug concentration as high as 0.5 mg/ml wasachieved by dissolving the API first in DMA at the concentration of 60mg/ml and then adding the DMA concentration in 50:50 TBA/citrate buffersolution. In order to minimize the residual solvent level in thefinished lyophilized drug product, a few approaches were considered:

-   -   1. Reducing the initial TBA amount may lead to less residual        TBA.    -   2. Reducing the initial DMA amount may lead to less residual        DMA.    -   3. Reducing the initial cyclodextrin amount may diminish the        solvent entrapment in this excipient.    -   4. Optimizing the freeze drying parameters may facilitate        removal of solvents in both sublimation and desorption        processes.

To reduce the initial TBA amount in the formulation, a small study wasconducted to evaluate the API solubility in varying levels of TBA in theDexolve-based lead formulations. API was dissolved in DMA at 50 mg/mland then added to the solution containing Dexolve, citrate buffer andTBA. Table 18 shows the formulation compositions in the solubilitystudy.

TABLE 18 Formulation XIV XV XVI API (mg/ml) 0.5 (added as a 50 mg/mlsolution in DMA) Dexolve-7 (mg/ml) 50 20 mM Citrate Buffer 80 70 60 (%v/v) TBA (% v/v) 20 30 40

It was observed that no clear solution was obtained when TBA level was30% v/v or less. This suggested that a minimum 35-40% v/v TBA isrequired to maintain a drug concentration at 0.5 mg/ml in the bulksolution. If DMA and/or Dexolve level is also reduced, an even higherlevel of TBA would be needed. There is little room to move the TBA leveldown from 50% v/v level used in the third screen. Therefore, it wasdecided to reduce the target drug concentration in the fourth screen inorder to achieve lower solvent content. Given a maximum fill volume of 8ml in a 20 cc vial and a drug loading of 1 mg/vial instead of 2 mg/vial,the lowest drug concentration to be formulated is 0.125 mg/ml.

The bulk solution compositions of the five formulations in the fourthscreen were described in Table 19.

TABLE 19 Formulation XXVII XXVIII XXIX XXX XXXI API (mg/ml) 0.125 0.1250.125 0.25 0.40 Dexolve-7 (mg/ml) 20 20 mM Citrate Buffer 100 75 75 7065 (% v/v) TBA (% v/v) n/a 25 25 30 35 DMA (% v/v) 0.25 0.25 n/a 0.500.79 Fill volume (ml/vial) 8.24 8.24 8.24 4.12 2.5 Total solid content203 194 194 74 68 (mg/vial)

Formulations XXVII, XXVIII and XXIX were all prepared at a drugconcentration of 0.125 mg/ml. Formulation XXVII contained no TBA whileFormulation XXVIII and XXIX contained 25% v/v TBA. Formulation XXIXcontained no DMA while Formulation XXVII and XXVIII both had the drugadded as a 50 mg/ml solution in DMA. Formulations XXX and XXXI hadrelatively higher levels of TBA at 30% v/v and 35% v/v, respectively. Asa result, they were able to achieve a higher drug concentration at 0.25mg/ml and 0.40 mg/ml, respectively. Based on the result from the firstscreen, the formulation containing Captisol® at 17 mg/ml obtained anacceptable cake structure. Therefore, Dexolve at a concentration of 20mg/ml instead of 50 mg/ml was used in the new screen to mitigate thesolvent entrapment tendency. In the lyophilization cycle of the fourthscreen, all the cycle parameters were kept the same as in the previousstudies except for the secondary drying. The shelf temperature in thesecondary drying step was increased from 25° C. to 40° C. and the dryingtime was prolonged from 6 hours to 12 hours.

All the five formulations obtained good freeze-dried cake materials,with Formulation XXVII providing the most elegant cake appearance. Afterbeing reconstituted with 4 ml D5W, drug precipitation was observed inRx30 vial within 60 minutes. The other formulations post reconstitutionstayed in clear solution for at least 3 hours based on visualobservation.

The weight loss of the lyophilized samples measured by TGA was reducedto a great extent as compared with previous lots. In correspondence, theresidual solvent level of each formulation detected by the GC method wasalso reduced, as shown in Table 20.

TABLE 20 Weight loss from Formulation TBA (mg/vial) DMA (mg/vial) TGA (%w/w) XXVII 0.05 11.34 1.55 XXVIII 8.81 8.18 XXIX 4.67 n/a 3.99 XXX 3.014.69 XXXI 2.25 2.86 2.17

Both the TBA level and the DMA level decreased with the decrease of thetotal solid content of the lyophilized cake. It was speculated that theless solid content led to the smaller cake thickness, which resulted inhigher heat transfer efficiency for the solvent to be removed from thecake. The tiny amount of TBA found in Formulation XXVII was suspected tocome from cross contamination. As the TBA-free formulation is the mostpreferable from the toxicology and regulatory perspective, FormulationXXVII was considered as the primary formulation to move forward in thenext stage of formulation development. Formulation XXXI was consideredas the backup along with Formulation XX, the mannitol-based formulation.During the formulation preparation, Dexolve revealed some quality issuessuch as the presence of unknown fibers and large colored particles.Besides, despite its comparable physical and chemical properties withCaptisol®, it has not been used in any FDA approved IV drug products,which is a potential regulatory barrier for its use in the clinicalstudy. Hence Captisol® and Kleptose® were used instead in the furtherdevelopment work.

Four formulations were selected out of the formulation screen as thelead candidates for the further process development. The fourformulations are shown in Table 21.

TABLE 21 Formulation Formulation Formulation Formulation IA IC II IIIAPI (mg/mL) * 0.125 0.125 0.40 0.50 Excipients Captisol ® Kleptose ®Captisol ® Mannitol (30 mg/mL) (30 mg/mL) (20 mg/mL) (50 mg/mL) Citratebuffer 100 100 60 50 (% v/v) TBA (% v/v) 0 0 40 50

The cyclodextrin levels of the two TBA-free formulations, FormulationsIA and IC, were increased from 20 mg/ml to 30 mg/ml to provide somesolubility margin as the DMA cosolvent level in the bulk solution was tobe further reduced in the process development. The other twoformulations, II and III, contained 40-50% TBA to accommodate a higherdrug loading than the TBA-free formulations.

Example 5: Thermal Analysis of Lyophilization Formulations

Prior to the process development work, a series of low temperaturethermal analysis work were conducted on each of the four leadformulations listed in Table 21 to characterize the physical andchemical behaviors of the formulations in a freeze drying process. Inthe electrical resistance (ER) measurement, the material was cooled andwarmed at an average controlled rate and the deviation in resistance wasused to determine an onset temperature of the phase transition uponwarming. In the freeze-drying microscope (FDM) measurement, the materialwas cooled and warmed in a sample cell under the temperature controlledfreeze drying stage. Changes in the frozen and dried portions of thesample during the phase transition were visually observed under themicroscope and the onset temperature was recorded. In the lowtemperature differential scanning calorimetry (LT-DSC) method, thesample was cooled to a complete freezing first, and then was warmed at amodulated heating rate. The glass transition event was detected in aresultant reversing heat flow. The minimum freezing temperature requiredfor complete solidification during freezing, the phase transitiontemperature upon warming, and the temperature at which the first void inthe frozen material was observed under FDM upon warming were identifiedand summarized in Table 22.

TABLE 22 Phase Glass Initial Void Freezing Freezing TransitionTransition Temp. upon Product Temp. Temp. Temp. Temp. warming Temp.(FDM) (ER) (ER) (LT-DSC) (FDM) Range Formulation IA −21.2° C. −26° C.−27° C. −36.6° C. −30° C. −32 to −34° C. Formulation IC −22.9° C. −12°C. −12° C. −20.4° C. −13.6° C. −16 to −18° C. Formulation III −22.1° C.−20° C. −20° C. −34.9° C. −41.2° C. −44 to (Exotherm) −46° C.Formulation III −21.2° C. −20° C. −17° C. −33.5° C. −32° C. −36 to(Endotherm) −38° C.

In general, the ER results are for reference only and not so specific asFDM or LT-DSC results. The visual results obtained from FDM areconsidered to be most representative to what occurs in the vial. Arecommended product temperature range during primary drying wasdetermined thereafter for complete sublimation with the retention of thecake structure and the absence of collapse. In some cases, the productmay still remain stable when the product temperature is slightly higherthan the glass transition temperature. Typically the product is kept2-3° C. below the initial void temperature, which is considered to berepresentative of the collapse temperature of the lyophilized cake, toprovide some safety margins. For the mannitol-based formulation III,LT-DSC results indicated an exothermic event in the 2° C./min scan whichwas not seen in the 10° C./min scan. This suggested that the event iswarming rate dependent. The formulation may benefit from an annealingstep prior to primary drying to allow efficient crystallization of thecrystalline bulking agent mannitol. The thermal analysis results,especially the recommended product temperature range for primary drying,were used as references in the subsequent process development work.

Example 6: Development of Lyophilization Process

A lyophilization process may consist of three stages: freezing, primarydrying, and secondary drying. A liquid formulation is transformed to alyophilized powder form by going through complete solidification throughfreezing stage, sublimation of ice and solvents through primary drying,and desorption of residual moisture and solvents through secondarydrying. The shelf temperature and chamber pressure in the primary dryingand secondary drying are the key process parameters which have greatimpact on the quality of the finished drug product. Five processdevelopment studies were executed to investigate the effect of each keyprocess parameter on the quality of the final lyophilized product. Aseries of tests were conducted on the finished drug product. The cakeappearance and structure was characterized by visual inspection. Thereconstituted solution was characterized by visual inspection and the pHmeasurement. The moisture content of the lyophilized cake was measuredby Karl Fisher method. The physico-chemical behaviors of the dried cakeat elevated temperatures were characterized by differential scanningcalorimetry (DSC) and thermogravimetic analysis (TGA). The residualsolvent level was quantified by gas chromotography (GC) method. Theassay and purity was measured by HPLC.

Example 7: Influence of Shelf Temperature in Primary Drying on FinishedProducts

The objective of this study was to evaluate the influence of the shelftemperature on the product temperature during primary drying. The shelftemperature was elevated from −34° C. to −16° C. stepwise with theconstant chamber pressure of 60 mTorr throughout primary drying. Thefour lead formulations described in Table 21 were compounded, filledinto 20 ml glass vials, and lyophilized with a target drug loading of 1mg/vial. The cycle parameters were displayed in Table 23.

TABLE 23 Shelf Temp. Soak Time Ramping Rate Pressure Step Setpoint (°C.) (hours) (° C./hour) Setpoint Product 5 2 30 Evac. To 12 Loading/psia to ensure Freezing chamber is airtight Freezing −50 3 30    60microns Annealing −18 3 30 Freezing −50 3 30 Primary −34 1.5 30 Drying−31 1.2 −28 1.0 −25 1.0 −22 0.8 −19 1.3 −16 77.6 Secondary 40 12.1Drying Stoppering 40 14.7 PSIA

During the primary drying, it was observed that the product temperatureof each formulation increased on average 0.8-1.4° C. per 3° increase inshelf temperature. The product temperature change was more prominent inFormulations II and III than in Formulations IA and IC. The productbreak temperatures, denoted as the product temperature at which the icesublimation was completed, were −35.9° C., −34.8° C., −40.7° C., and−40.1° C. for Formulations IA, IC, II and III, respectively. The breaktemperatures of all the formulations except for Formulation II werebelow the recommended product temperature ranges obtained from the lowtemperature thermal analysis. This suggested that collapse could haveoccurred in Formulation II while the other three formulations attainedgood retention of the cake structure.

The finished products of the four sublots showed acceptable cakeappearance with varying degrees of shrinkage. The assay of each sublotwas in an acceptable range of 95-105%. The residual moistures of thefour sublots were all less than 0.2%. The lyophilized material wasreconstituted with purified water at a volume of 2, 4, and 8 ml.Formulations IA, IC and II rendered a clear and colorless solution whichstayed physically stable for 4 hours by visual inspection. FormulationIII was cloudy and would require alternative diluent containing organicsolvent. The pH of each reconstituted solution was in the range of4.5-4.9. The residual solvent level of each sublot was quantified by GCand listed in Table 24.

TABLE 24 Formulation Formulation Formulation IA Formulation IC II IIIResidual 7.64 6.37 4.74 0.58 DMA (mg/vial) Residual 0.04 0.05 2.11 0.07TBA (mg/vial)

According to the ICH guidance, DMA was considered as Class 2 solvent andthe maximum daily uptake was set as 10.9 mg/day. This guidance does notapply to potential new drug products used during the clinical researchstages of development. Hence the stated DMA limit is only used as abenchmark. TBA is not listed in the ICH guidance. The maximum dailyuptake of residual TBA was set as 0.15 mg/day. Given a top dose of 2mg/day, the residual DMA and TBA in finished products are expected to bebelow 5.45 mg/vial and 0.075 mg/vial respectively. All the formulationsexcept for Formulation III exceeded the residual solvent limit.Therefore, process optimization was conducted to further reduce theresidual solvent.

Example 8: Influence of Chamber Pressure in Primary Drying on FinishedProducts

This study was designed to evaluate the influence of the chamberpressure on the product temperature during primary drying. The chamberpressure was elevated from 40 mTorr to 200 mTorr stepwise with theconstant shelf temperature at −34° C. throughout primary drying. Thecycle parameters were described in table 25.

TABLE 25 Shelf Temp. Soak Ramping Setpoint Time Rate Step (° C.) (hours)(° C./hour) Pressure Setpoint Product 5 2 Evac. To 12 psia Loading/ 30to ensure chamber Freezing is airtight Freezing −50 3 30 Annealing −18 330 Freezing −50 3 30 40 microns Primary Drying −34 5.0 50 microns 0.8 60microns 1.0 70 microns 1.2 80 microns 0.3 90 microns 21.5 100 microns25.4 120 microns 16.8 140 microns 5.6 160 microns 27.5 200 microns 30200 microns Secondary 40 12.0 200 microns Drying Stoppering 40 14.7 PSIA

Four sublots were formulated in this study. Formulations IA, III and IIIwere the same as illustrated in Table 21. The only difference from theprocess study Run #1 was that, the starting concentration of API in DMAwas increased from 75 mg/ml to 120 mg/ml to lower the initial amount ofDMA in the bulk solution. Formulation IC was not evaluated in this studyas it was theorized that Formulation IA containing Captisol® maydemonstrate comparable physical and chemical behaviors as FormulationIC, the Kleptose®-based formulation. Instead, Rx4, a Captisol® based,solvent-free formulation was added to the study plan to assess thefeasibility of compounding the bulk solution with no aid of TBA or DMA.The formulation dissolves 0.125 mg/ml Form C directly in the same 20 mMcitrate buffer solution with 300 mg/ml Captisol®. Drug precipitation wasobserved in Rx4 during the compounding process and the precipitated drugparticles were filtered out prior to the lyophilization. Later a lowassay value of 28.8% in the lyophilized sample of Rx4 sublot confirmedthat the solvent-free formulation is not a viable option even with tenfolds higher cyclodextrin to facilitate solubilization of the drug.Therefore Rx4 was not considered in the further studies.

During the primary drying, it was observed that the product temperatureof the sublot Formulation IA, 2, and 3 increased on average 1.1° C. per10 micron increase in the chamber pressure from 50 to 70 microns. From80 to 140 microns, product temperature increased approximately 0.5° C.for each 10 micron increment. The product break temperatures were −36.7°C. and −37.7° C. for Rx2 and 3 respectively. The break temperature ofRx3 was within the recommended product temperature range, suggestingthat Rx3 sublot was freeze dried with retention and absence of collapse.In contrast, the product break temperature of Rx2 was higher than therecommended product temperature range. Obvious collapse was observed ina few Rx2 sample vials. Formulation IA did not undergo a breaktemperature implying incomplete sublimation in these sublot vials.

The finished products of the four sublots showed acceptable cakeappearance with varying degrees of shrinkage. The assay of the threesublot Formulation IA, II, and III were all in an acceptable range of95-100%. The moisture content and the reconstitution results of thesublots Formulation IA, II and III in the process Run #2 were similar tothose obtained in the process Run #1. The residual solvent level of eachsublot was quantified by GC and listed in Table 26.

TABLE 26 Formulation Formulation Formulation Formulation IA IC II IIIResidual DMA 3.71 NA 2.21 0.4 (mg/vial) Residual TBA 0.04 NA 1.63 0.03(mg/vial)

The reduced residual solvent levels of all the sublots in this studyindicated that decreasing the initial amount of DMA charge in the bulksolution is an effective approach to minimize the residual DMA level inthe finished drug product.

Example 9: Influence of Chamber Pressure in Secondary Drying on FinishedProducts

This study was designed to evaluate the influence of a higher chamberpressure in the secondary drying on the residual solvent level of thefinished drug products. Three formulation sublots, Formulation IC, II,and III were evaluated in this study. The compositions of eachformulation were the same as listed in Table 21. The API was dissolvedin DMA at a concentration of 120 mg/ml in all the three formulations inthis study. Formulation IA was abandoned in this study as Kleptose® wasconsidered advantageous over Captisol® due to similar chemicalproperties and lower material cost at this point. Based on the producttemperature profiles derived from the previous two process studies, theshelf temperature and the chamber pressure of the primary drying in thisstudy were set at −22° C. and 40 microns respectively in order to yielda product temperature range of −40° C. to −42° C. This conservativesetting was to ensure all the three formulations retain the structurewith absence of collapse during the primary drying. The chamber pressureof the secondary drying was increased from 40 microns to 600 microns inthis study. It was expected that a higher chamber pressure would createa more nitrogen rich environment and more efficient heat transfer whichmay aid in desorption of residual solvents. The cycle parameters weredescribed in Table 27.

TABLE 27 Shelf Temp. Soak Ramping Setpoint Time Rate Step (° C.) (hours)(° C./hour) Pressure Setpoint Product 5 2 Evac. To 12 psia to Loading/30 ensure chamber is Freezing airtight Freezing −50 3 30 Annealing −18 330 Freezing −50 3 30 40 microns Primary −22 111 40 microns Drying 4 600microns 30 600 microns Secondary 40 12 600 microns Drying Stoppering 4014.7 PSIA

In this study, the product break temperatures of Formulation IC rangedbetween −35.2° C. and −38.2° C., much lower than the recommended producttemperature range of −16° C. to −18° C. This implied that there is stilla lot of room in the further lyophilization cycle optimization. Theproduct break temperatures of Formulations II and III were −38° C. and−39° C. respectively. The break temperatures of Formulation II exceededthe recommended product temperature range, suggesting that a moreconservative cycle would be needed for this formulation to avoid thecollapse.

The finished products of the three sublots showed acceptable cakeappearance with varying degrees of shrinkage. The moisture content andthe reconstitution results of the sublots IC, II and III in the processRun #3 were similar to the results in the previous studies. The residualsolvent level of each sublot was quantified by GC and listed in Table28.

TABLE 28 Formulation Formulation Formulation Formulation IA IC II IIIResidual DMA NA 6.22 2.7 0.62 (mg/vial) Residual TBA NA 0.11 3.3 0.05(mg/vial)

It showed that the residual DMA level in Formulation IC was still higherthan the desired upper limit. Increase in the chamber pressure of thesecondary drying had minimal effect on the solvent desorption.

Example 10: Influence of Shelf Temperature and Drying Time in SecondaryDrying on Finished Products

This study was designed to evaluate the influences of the shelftemperature and the drying time in the secondary drying on the residualsolvent level of the finished drug product. Formulation IC was selectedas the lead formulation to proceed due to its superior reconstitutionperformance. Two other variations, Formulations IC, XXXIII and XXXV,were added in this study to evaluate the influences of the excipients onthe residual solvent. Formulation XXXIII differed from IC with a lowerKleptose® level of 20 mg/ml. The intent was to evaluate whether reducingKleptose® concentration would help reduce the residual solvent. Aprevious solubility study showed that for Formulation IC, the minimumKleptose® concentration was 25 mg/ml to assure complete drug dissolutionin the bulk solution. Therefore when the Kleptose® concentration wasreduced to 20 mg/ml in Formulation XXXIII sublot, the API was dissolvedin DMA at 60 mg/ml instead of 120 mg/ml to ensure complete drugdissolution in the bulk solution. Formulation XXXV differed from IC withthe addition of 40 mg/ml mannitol. The intent was to evaluate whetherpresence of mannitol providing a more crystalline structure in thelyophilized cake would promote the removal of the residual solvent. Thecompositions of the three formulations are listed in Table 29.

TABLE 29 Formulation Formulation Formulation IC XXXIII XXXV API (mg/mL)0.125 0.125 (added as 0.125 (added as (added as 60 mg/ml in 120 mg/ml in120 mg/ml in DMA) DMA) DMA) Excipients Kleptose ® Kleptose ® Kleptose ®(30 mg/mL) (20 mg/mL) (30 mg/mL) Mannitol (50 mg/mL) Solvents pH 4.5citric Buffer (100% v/v)

The shelf temperature and the chamber pressure of the primary drying inthis study were set at −28° C. and 60 microns respectively to provide aconservative lyophilization cycle for all the formulations. An annealingstep was added during the freezing stage to facilitate thecrystallization of mannitol in Formulation XXXV. Secondary drying wasconducted at an increased shelf temperature of 50° C. for 12, 18, and 24hours. Sample vials were pulled out of the lyophilizer at each timepoint to check the residual solvent level change over time. The cycleparameters were described in Table 30.

TABLE 30 Shelf Temp. Soak Ramping Setpoint Time Rate Step (° C.) (hours)(° C./hour) Pressure Setpoint Product 5 2 Evac. To 12 psia to Loading/30 ensure chamber is Freezing Freezing −50 3 airtight 30 Annealing −18 330 Freezing −50 3 30 60 microns Primary −28 104  60 microns Drying 30 60microns Secondary 50 12, 18, 24 60 microns Drying Stoppering 50 14.7PSIA

The product break temperature ranged from −33° C. to −36° C. in primarydrying, which was way below the recommended product temperature ofFormulation IC from LT-TA. This suggested that the currentlyophilization cycle was too conservative and there was still room toincrease the shelf temperature and/or the chamber pressure in primarydrying to shorten the drying time in further development. The finishedproducts of the three sublots showed acceptable cake appearance withvarying degrees of shrinkage. The residual solvent level of each sublotwas quantified by GC and listed in Table 31.

TABLE 31 Formulation Formulation Formulation IC XXXIII XXXV Drying time(mg/vial) (mg/vial) (mg/vial) 12 hr 5.8 11.2 5.6 18 hr 5.7 11.2 5.8 24hr 5.7 11.2 5.5

It showed that the residual DMA level in Formulation IC was reducedslightly from 6.2 mg/vial in Run #3 to 5.8 mg/vial. Prolonged dryingtime beyond 12 hour has no impact on the removal of the residualsolvent. Formulation XXXIII showed the highest residual DMA level amongthe three sublots mainly because its initial DMA charge was doubled theamount in the other two formulations. Formulation XXXV had similarresidual DMA level as Formulation IC, indicating that addition ofmannitol had little impact on the removal of the residual DMA. As theresult showed a strong correlation between the initial DMA charge to thebulk solution and the residual DMA in the lyophilized cake, it wasdecided to further reduce the initial DMA charge in the formulation inthe next study in order to bring down the residual solvent level.

Example 11: Refinement of the Formulation and Lyophilization ProcessParameters

This study was designed to refine the formulation and process parametersof the lead formulation IC based on the previous process study results.First of all, a quick solubility study was conducted to evaluate themaximum feasible concentration of API in DMA. When API was dissolved inDMA at 150 mg/ml and added to the bulk solution, undissolved particleswere observed prior to filtration and drug precipitation occurred in thebulk solution after overnight storage. When API was dissolved in DMA at135 mg/ml and added to the bulk solution, the clear and colorlesssolution was obtained and remained stable after overnight storage.Therefore the API in DMA solution concentration was increased from 120mg/ml in the last study to 135 mg/ml in the new formulation ID. Thecompositions of the other ingredients in Formulation ID remained thesame as in Formulation IC and were described in Table 32.

TABLE 32 Formulation ID API (mg/mL) 0.125 (added as 135 mg/ml in DMA)Excipients Kleptose ® (30 mg/mL) Solvents pH 4.3 citrate Buffer

The target pH value of the citrate buffer was adjusted from 4.5 to 4.3to ensure more robust solution stability below pH 4.5. The subsequentHPLC tests confirmed that both the assay and the purity of the filteredbulk solution remained stable with no obvious degradation growth within8 hours (data not shown). Therefore the recommended holding time for thebulk solution upon preparation is 8 hours at ambient condition.

In the lyophilization cycle, the shelf temperature and the chamberpressure of primary drying were elevated to −16° C. and 140 micronsrespectively to enhance the sublimation rate. The more aggressiveprocess parameters reduced the primary drying time from over 100 hoursin the previous study to about 60 hours. To add some safety margin, thefinal primary drying time was set at 70 hours. Secondary drying wasprocessed at 50° C. and 140 microns for 12 hours. The cycle parametersare described in Table 33.

TABLE 33 Shelf Temp. Soak Ramping Setpoint Time Rate Step (° C.) (hours)(° C./hour) Pressure Setpoint Product 5 2 Evac. To 12 psia toLoading/Freezing 30 ensure chamber is Freezing −50 3 airtight 30 PrimaryDrying −16 70 140 microns 30 140 microns Secondary Drying 50 12 140microns Stoppering 50 14.7 PSIA

The temperature profile of the lyophilization cycle is illustrated inFIG. 31.

Product break temperature ranges from −28° C. to −30° C. during primarydrying suggesting that the product was dried with absence of collapseand there is still room to improve the primary drying rate in furtherdevelopment.

The finished product of Formulation ID showed dense and uniform cakeappearance. The moisture content is below the detection limit of theKarl Fisher method. The residual DMA level dropped to 4.2 mg/vial, whichwas below the target upper limit of 5.45 mg/vial. The resulting assayvalue was unexpectedly high at 110.8%. Compounding the API in DMA byvolume was identified to be the high risk step contributing to the highassay. It was suggested to compound by weight in lieu of volume infuture studies. The reconstitution performance was comparable toprevious Formulation IC samples. Overall, the results of the finishedproduct testing were considered to be acceptable for the FIH clinicalstudy.

Summary of Process Development

Five process studies were executed in series to investigate the impactof the critical process parameters on the quality of finished drugproducts, especially the residual solvent content. The refinement of theformulation was conducted concurrently with the process development. Itwas found that presence of cyclodextrin in the formulation led toentrapment of the residual DMA in the dried cake. Secondary drying hadminimal impact on the removal of the residual DMA. Reducing initial DMAcharge in the formulation and using more aggressive cycle parameters inprimary drying helped to reduce the residual DMA. The formulation ID andthe lyophilization process identified in the last process study weretaken to the scale up demo batch for evaluation. A complete processdiagram including compounding, freeze-drying, filtration, filling, andpackaging was illustrated in FIG. 32.

Example 12: Stability of Finished Drug Products

Preliminary stability studies have been performed during the formulationscreen. Among all the prototype formulations, Formulation IX compositionis the closest to the FIH formulation. Formulation IC which wasevaluated towards the end of the process development has exactly thesame composition as the FIH formulation except for the residual solventlevel. Table 34 compares the formulation compositions of IX and IC asopposed to the FIH formulation.

TABLE 34 Formulation Formulation Formulation ID Lot No. IX IC (FIH) FormC (mg/vial) 0.76 1.0 1.0 Citric acid anhydrous, USP 6.1 17.7 17.7(mg/vial) Sodium citrate anhydrous, 8.2 17.6 17.6 USP (mg/vial)Kleptose ® HPB, 67 240 240 parenteral grade (mg/vial) TBA (in processmedia) Removed 0 0 upon drying DMA, PW (in process Removed upon dryingmedia)* Total 82.1 276.3 276.3

The stability data of Formulation IX and Formulation IC is presented inTable 35.

TABLE 35 Purity Assay (% area) (% label claim) Formulation No. IX IC IXIC Initial 98.5 97.8 93.0 101.0 1 mo @ 40° C./75% 98.6 97.9 96.3 104.6RH 3 mo @ 40° C./75% 99.6 / 96.0 / RH 1 mo @ 25° C./60% 98.6 / 96.5 / RH3 mo @ 25° C./60% 99.6 / 95.8 / RH

Formulation IX samples remained stable at the accelerated condition of40° C./75% RH for three months with no obvious degradant growth.Similarly, Formulation IC samples remained stable at the acceleratedcondition of 40° C./75% RH for one month. The accelerated stability dataobtained so far showed very promising results indicating that finisheddrug products could have an acceptable shelf life under room temperaturestorage condition.

Example 13: In-use Stability of Reconstituted Solutions

Reconstitution studies were conducted with either D5W or purified waterwith different volumes from 2 ml to 8 ml. Similar reconstitutionperformance was observed regardless of the type or the volume of thediluents. Osmolality measurement was conducted on each reconstitutedsolution and the results are shown in Table 36.

TABLE 36 Diluent volume 2 ml 8 ml D5W 636 ± 2 mOsm/kg 404 ± 1 mOsm/kgPurified water 283 ± 0 mOsm/kg  72 ± 1 mOsm/kg Water For Injection 301 ±0 mOsm/kg /

It was found that reconstitution with 2 ml purified water rendered anosmolality of 283 mOsm/kg. This value is very close to the human plasmaosmolality of 285-295 mOsm/kg, while the other three reconstitutedsolutions exhibited very distinct osmolality values. Subsequently thesame measurement was repeated with 2 ml water for injection (WFI) andthe osmolality value of 301 mOsm/kg was obtained. As a result, 2 mlwater for injection was recommended as the reconstitution diluent due toits physiologically isotonic characteristic and used in the followingreconstitution studies to evaluate the in-use stability of thereconstituted solution. The assay and purity of the reconstitutedsolution were measured by HPLC every two hours for 8 hours. The resultsare shown in Table 37.

TABLE 37 Hydrolysis Hydrolysis Assay Purity Degradant 1 Degradant 2 TimePoint (% LC) (% area) (% area) (% area) T = 0 108.8 97.90 0.16 0.39 T =2 hr 109.1 97.91 0.16 0.39 T = 4 hr 108.4 97.91 0.16 0.39 T = 6 hr 108.497.90 0.16 0.40 T = 8 hr 108.5 97.89 0.17 0.40

The in-use stability data demonstrated that the formulation solutionupon reconstitution remained stable for 8 hours at room temperaturecondition. Meanwhile no drug precipitation was observed after overnightstorage at room temperature condition by visual inspection, assuring thephysical stability of the reconstituted solution.

Based on the aforementioned stability results, the proposedreconstitution procedures are described as follows:

Reconstitute each vial with 2 mL sterile water for injection. Gentlyshake or roll the vial until all solids are dissolved. The resultingsolution will contain Form C 0.50 mg/mL. The solution should be clearand colorless. The reconstituted solution remains stable in the vial atroom temperature for 8 hours. Inspect the solution visually forparticulate matter and discoloration prior to administration. Withdrawthe required amount of Form C solution to deliver the desired dose.

The embodiments described above are intended to be merely exemplary, andthose skilled in the art will recognize, or will be able to ascertainusing no more than routine experimentation, numerous equivalents ofspecific compounds, materials, and procedures. All such equivalents areconsidered to be within the scope of the invention and are encompassedby the appended claims.

Formulation of the invention are for use in medicine.

Formulations of the invention are for use in the methods of treatmentprovided herein.

What is claimed:
 1. A method of treating a cancer comprisingadministering to a mammal having cancer a lyophilized formulationcomprising: Compound 1(2-(4-chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamide),or a stereoisomer or mixture of stereoisomers, pharmaceuticallyacceptable salt, tautomer, prodrug, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, a buffer and a bulking agent, whereinthe cancer is a solid tumor and the method further comprisesadministering calcium, calcitriol, and vitamin D supplementation.
 2. Themethod of claim 1, further comprising administering a therapeuticallyeffective amount of a second active agent or a support care therapy. 3.The method of claim 2, wherein the second active agent is 1) atherapeutic antibody that specifically binds to a cancer antigen or apharmacologically active mutant thereof, 2) a hematopoietic growthfactor, 3) a cytokine, 4) an anti-cancer agent, 5) an antibiotic, 6) acox-2 inhibitor, 7) an immunomodulatory agent, 8) an immunosuppressiveagent, or 9) a corticosteroid.
 4. A method of treating a cancercomprising administering to a mammal having cancer a reconstitutedformulation obtained from a lyophilized formulation and a diluent,wherein the lyophilized formulation comprises: Compound 1(2-(4-chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamide),or a stereoisomer or mixture of stereoisomers, pharmaceuticallyacceptable salt, tautomer, prodrug, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, a buffer and a bulking agent, whereinthe cancer is a solid tumor and the method further comprisesadministering calcium, calcitriol, and vitamin D supplementation.
 5. Themethod of claim 4, wherein the method comprises administering theformulation intravenously.
 6. The method of claim 4, further comprisingadministering a therapeutically effective amount of a second activeagent or a support care therapy.
 7. The method of claim 6, wherein thesecond active agent is 1) a therapeutic antibody that specifically bindsto a cancer antigen or a pharmacologically active mutant thereof, 2) ahematopoietic growth factor, 3) a cytokine, 4) an anti-cancer agent, 5)an antibiotic, 6) a cox-2 inhibitor, 7) an immunomodulatory agent, 8) animmunosuppressive agent, or 9) a corticosteroid.
 8. The method of claim1, wherein the cancer is a cancer of bladder, bone, brain, breast,cervix, chest, colon, endrometrium, esophagus, eye, head, kidney, liver,lymph nodes, lung, mouth, neck, ovaries, pancreas, prostate, rectum,stomach, testis, throat, or uterus.
 9. The method of claim 1 comprisingadministering calcium, calcitriol, and vitamin D supplementation withthe lyophilized formulation.
 10. The method of claim 1 comprisingadministering calcium, calcitriol, and vitamin D supplementation priorto the lyophilized formulation.
 11. The method of claim 4, wherein thecancer is a cancer of bladder, bone, brain, breast, cervix, chest,colon, endrometrium, esophagus, eye, head, kidney, liver, lymph nodes,lung, mouth, neck, ovaries, pancreas, prostate, rectum, stomach, testis,throat, or uterus.
 12. The method of claim 4 comprising administeringcalcium, calcitriol, and vitamin D supplementation with thereconstituted formulation.
 13. The method of claim 4 comprisingadministering calcium, calcitriol, and vitamin D supplementation priorto the reconstituted formulation.